Compositions comprising promoter sequences and methods of use

ABSTRACT

Nucleic acid molecules, fragments and variants thereof having promoter activity are provided in the current invention. The invention also provides vectors containing a nucleic acid molecule of the invention and cells comprising the vectors. Methods for making and using the nucleic acid molecules of the invention are further provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/330,471, filed Jan. 12, 2006, which claims the benefit of U.S.Provisional Application No. 60/644,189, filed Jan. 14, 2005, which isherein incorporated by reference in its entirety.

INCORPORATION OF SEQUENCE LISTING

The application contains a sequence listing, a paper copy of which isfiled concurrently herewith via EFS Web. The CRF copy of the sequencelisting filed on Apr. 7, 2006 has been transferred from the parentapplication (U.S. application Ser. No. 11/330,471). The content of theattached paper copy of the listing and the CRF copy transferred fromU.S. application Ser. No. 11/330,471 are the same. The sequence listingis hereby incorporated in its entirety into the instant specification.

FIELD OF THE INVENTION

The present invention is directed to promoters in general, as well as,nucleic acid constructs comprising such promoters operably associatedwith a nucleic acid of interest in a recombinant nucleic acid molecule,cells containing the same, and methods of making and using the same.

BACKGROUND OF THE INVENTION

The gastrointestinal tract is the most densely colonized region of thehuman body (Savage, Ann. Rev. Microbiol. 31, 107 (1977); Tannock, Normalmicroflora (Chapman and Hall, London 1995)) and the accumulated evidenceindicates that this collection of microbes has a powerful influence onthe host in which it resides. Comparisons between germ free andconventional animals have shown that many biochemical, physiological andimmunological functions are influenced by the presence of the diverseand metabolically active bacterial community residing in thegastrointestinal tract (Marteau and Rambaud, FEMS Microbiol. Rev. 12,207 (1993); Norin et al., Appl. Environ. Microbiol. 74, 1850 (1991);Tannock, supra). Lactobacilli are important residents of the microflora(Ahrne et al., J. Appl. Microbiol. 85, 88 (1998); Kimura et al., Appl.Environ. Microbiol. 63, 3394 (1997)), and have been the subject ofintense and growing interest because of their possible role in themaintenance of gastrointestinal health (Bengmark, Gut 42, 2 (1998)). Ofimmense importance to lactobacilli functioning in this role is theability to endure in the harsh conditions of the gastrointestinal tract,where the gastric pH frequently falls below 2.0 in healthy individuals(McLauchlan et al., Gut 30, 573 (1998)).

The identification of conditionally expressed genes provides a wealth ofinsight into the physiological consequences of and responses to a givenstimulus. In the case of Lactobacillus acidophilus, a significantchallenge has been in understanding the intestinal roles and activitiesof this organism. An important element in this regard is thedetermination of which characteristics are important for the survivaland success of this organism in the gastrointestinal tract. Whiledifferential display (Liang and Pardee, Science 257, 967 (1992); Welshet al., Nucleic Acids Res. 20, 4965 (1992)) has been used extensively toidentify conditionally expressed genes in eukaryotes, the application ofthis methodology in prokaryotes has not been explored to a comparativelysignificant extent (Abu Kwaik and Pederson, Mol. Microbiol. 21, 543(1996); Fislage, Electrophoresis 19, 613 (1998); Fislage et al., NucleicAcids Res. 25, 1830 (1997); Wong and McClelland, Proc. Natl. Acad. Sci.USA 91, 639 (1994); Zhang and Normark, Science 273, 1234 (1996)). Someof the practical problems in employing these methods in prokaryotesinclude the relatively large proportion of structural RNA species in thetotal RNA, the low level of polyadenylation of mRNA (Sarkar, Ann. Rev.Biochem. 66, 173 (1997)), which prohibits the use of 3′ dT anchoredprimers and the structural instability and short half life of lowabundance mRNA species of prokaryotes as compared to eukaryotes (Higginset al., Curr. Opin. Genet. Dev. 2:739 (1992)).

The present invention contributes to the art by providing promoters ascompositions and for use in methods of expressing nucleic acids in avariety of conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the experimental design of the microarrayassays described herein.

FIG. 2 provides an overview of expression data from a GUS reporter geneassay. pFOS refers to the 502_sugar promoter sequence set forth in SEQID NO: 72; pTRE refers to the 1012_sugar promoter sequence set forth inSEQ ID NO:73; and, pPGM refers to the 185_high promoter set forth in SEQID NO:6.

FIG. 3 is a detailed representation (through time) of the pFOS (promoter502_sugars) (SEQ ID NO:72) data. It shows that this promoter isinducible in the presence of FOS when compared to glucose and fructose.

FIG. 4 provides a non-limiting schematic of an expression vector for thepFOS promoter (SEQ ID NO:72).

FIG. 5 provides a non-limiting schematic of an expression vector for thepTRE promoter (SEQ ID NO:73).

FIG. 6 provides a non-limiting schematic of an expression vector for thepPGM promoter (SEQ ID NO:6).

SUMMARY OF THE INVENTION

Methods and compositions for regulating gene expression are provided.

Compositions comprise isolated nucleic acid molecules comprising (a) anucleic acid comprising a nucleotide sequence as set forth in any one ofSEQ ID NOS: 1-80 or a fragment thereof; (b) a nucleic acid thathybridizes to the complement of the nucleic acid of (a) under stringentconditions, wherein the sequence has promoter activity; and (c) anucleic acid having at least 70%, 80%, 90%, 95% or greater sequenceidentity to the nucleotide sequence set forth in any one of SEQ IDNOS:1-80, wherein the sequence has promoter activity.

Further provided are recombinant nucleic acid molecules of SEQ ID NOS:1-80 or biologically active variants or fragments thereof, wherein themolecules are operably linked to a heterologous nucleic acid ofinterest. Vectors having such recombinant nucleic acid molecules arealso provided, as are cells having a heterologous nucleic acid moleculecomprising the sequence of SEQ ID NOS:1-80 and biologically activevariants thereof.

Further provided are methods for controlling the transcription of anucleic acid of interest. One method comprises (a) providing ormaintaining the cell under non-inducing conditions, wherein the cellcomprises at least one of the recombinant nucleic acid molecules of anyone of SEQ ID NOS:1-80 or a biologically active variant or fragmentthereof or a vector having the same, and (b) subjecting the cell toinducing conditions whereby transcription of the nucleic acid ofinterest is increased as compared to the level of transcription of thenucleic acid of interest under non-inducing conditions. Inducingconditions can be produced by increasing or decreasing the pH of thecell relative to the pH of the cell under non-inducing conditions; byadministering or delivering the cell to a body cavity of the subject,wherein the body cavity has an acidic pH environment; by thefermentative production of an acid by the cell in a cell culture; by anincrease or decrease in temperature of the cell relative to thetemperature of the cell under non-inducing conditions; by an increase ordecrease in the concentration of a sugar in the cell relative to theconcentration of the sugar in the cell under non-inducing conditions;or, by the presence of a stress response protein.

Further included is a method to express a nucleotide sequence ofinterest in a cell comprising introducing into the cell a heterologousnucleic acid molecule comprising any one of SEQ ID NOS:1-80 or abiologically active variant or fragment thereof, wherein the nucleicacid molecule is operably linked to a nucleotide sequence of interest.

The foregoing and other objects and aspects of the invention aredescribed herein and the specification set forth below.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The present invention provides isolated nucleic acid moleculescomprising, consisting essentially of and/or consisting of thenucleotide sequences as set forth in SEQ ID NOS:1-80. Also provided areisolated nucleic acid molecules having promoter activity, wherein thenucleic acid molecule is selected from the group consisting of: (a) anucleic acid molecule comprising, consisting essentially of, and/orconsisting of a nucleotide sequence as set forth in SEQ ID NOS:1-80 or afragment thereof; (b) a nucleic acid molecule that hybridizes to thecomplement of the nucleic acid molecule of (a) under stringentconditions and has promoter activity; and (c) a nucleic acid moleculehaving at least 70%, 80%, 90%, 95% or greater sequence identity to thenucleic acid molecule of (a) or (b) and has promoter activity. A nucleicacid molecule having a nucleotide sequence that is complementary to anyone of the nucleic acid molecules described herein is also provided inthis invention.

In other embodiments, the present invention provides isolated nucleicacid molecules comprising the nucleotide sequences as set forth in SEQID NOS: 6, 72, or 73. Also provided are isolated nucleic acid moleculeshaving promoter activity, wherein the nucleic acid molecule is selectedfrom the group consisting of: (a) a nucleic acid molecule comprising,consisting essentially of, and/or consisting of a nucleotide sequence asset forth in SEQ ID NOS: 6, 72, or 73 or a fragment thereof; (b) anucleic acid molecule that hybridizes to the complement of the nucleicacid molecule of (a) under stringent conditions and has promoteractivity; and (c) a nucleic acid molecule having at least 70%, 80%, 90%,95% or greater sequence identity to the nucleic acid molecule of (a) or(b) and has promoter activity.

Variant nucleic acid molecules sufficiently identical to the nucleotidesequences set forth herein are also encompassed by the presentinvention. Additionally, fragments and sufficiently identical fragmentsof the nucleotide sequences are encompassed. Nucleotide sequences thatare complementary to a nucleotide sequence of the invention, and/or thathybridize to a nucleotide sequence, or complement thereof, of theinvention are also encompassed.

Compositions of this invention further include vectors and cells forrecombinant expression of the nucleic acid molecules described herein,as well as transgenic microbial and/or cell populations comprising thenucleic acids and/or vectors. Also included in the invention are methodsfor the recombinant production of heterologous peptides and/orpolypeptides and methods for their use.

Another aspect of the present invention is an isolated nucleic acidcomprising: (a) a first nucleotide sequence having promoter activity,wherein the promoter can be a constitutively active promoter or aninducible promoter, wherein the latter can be induced by a variety offactors, including but not limited to, pH, growth temperature, oxygencontent, a temperature shift, the composition of the growth medium(including the ionic strength/NaCl content), the presence or absence ofessential cell constituents or precursors, the growth phase and/or thegrowth rate of a cell or cell population, and any of a variety ofinducing compounds and/or chemicals that are well known in the art, asdescribed herein; and (b) a second nucleotide sequence having aposition, orientation, presence and/or sequence which imparts aregulatory effect on the expression of a nucleic acid sequence operablylinked to the first nucleotide sequence having promoter activity. Anucleic acid molecule of this embodiment can be, for example, a nucleicacid having a nucleotide sequence as set forth in SEQ ID NOS:1-80 or SEQID NO: 6, 72, or 73 as provided herein, and/or a nucleic acid thathybridizes with the complement of a nucleic acid having the nucleotidesequence as set forth in SEQ ID NOS:1-80 or SEQ ID NO:6, 72, or 73 andhas the promoter and regulatory activity described herein. The nucleicacid molecule of this embodiment can also be a nucleic acid moleculehaving at least 70% homology to a nucleic acid molecule having anucleotide sequence of SEQ ID NOS:1-80 or SEQ ID NO: 6, 72, or 73 andhaving promoter and regulatory activity as described herein.

In one embodiment, the nucleic acid molecule according to the presentinvention may be induced by sugar (including, but not limited to,glucose, fructose, sucrose, trehalose, fructooligosaccharide, raffinose,lactose and/or galactose) and may be referred to herein as a“sugar-induced” promoter. Suitably at least SEQ ID NOS:70-80 may besugar-induced promoters.

In another embodiment, the nucleic acid molecule according to thepresent invention may be induced by exposure to a stress response(including, but not limited to, change in pH, exposure to bile, oxalateand/or ethanol alone or in various combinations) or to a stress responseprotein and may be referred to herein as a “stress-induced” promoter.Suitably at least SEQ ID NOS:44-69 may be stress-induced promoters. Inother embodiments, exposure to a stress response contributes torepression of a promoter.

In another embodiment, the nucleic acid molecule according to thepresent invention may be induced by growth temperature or a shift intemperature (and may be referred to herein as a “temperature-induced”promoter).

In another embodiment, the nucleic acid molecule according to thepresent invention may be induced by Fos (and may be referred to hereinas a “Fos-induced” promoter). Suitably at least SEQ ID NO: 72.

The nucleic acid molecules comprising promoters of the present inventionhave applications in a number of scenarios. The promoters of thisinvention can be used for the expression of nucleic acid molecules toyield gene products, for example, during the normal course offermentation by cells such as bacterial cells, particularly lactic acidbacteria, in dairy, meat, vegetable, cereal, and other bioconversions.The promoters of this invention can also be used for the production ofgene products upon exposure of lactic acid bacteria to certainenvironmental stimuli (e.g., acid environments), including, for example,suspension into acidified foods or entry into the gastrointestinal tractor other body cavities as probiotic bacteria.

The nucleic acid molecules of this invention can be used in someembodiments for the expression of nucleic acid molecules encodingenzymes, antigens, proteins, peptides, etc., from lactic acid and/orother bacteria that can be used, for example, as delivery or productionsystems.

Accordingly, a further aspect of the invention is a recombinant nucleicacid comprising a promoter of this invention operably associated with anucleic acid of interest. In some embodiments, the nucleic acid ofinterest can encode a protein or peptide, the production of which can beupregulated, e.g., upon induction of the promoter. In other embodiments,the nucleic acid of interest can encode an antisense oligonucleotidethat can suppress or inhibit the production of a protein in a cell,e.g., upon induction of the promoter. In other embodiments, the nucleicacid of interest can encode a ribozyme, an interfering RNA (RNAi), etc.,that would be useful, for example, in situations where regulation ofgene expression and/or protein production is desired.

As noted above, in some embodiments, the nucleic acid of interest canencode an antisense RNA. In general, “antisense” refers to the use ofsmall, synthetic oligonucleotides to inhibit protein production byinhibiting the function of the target mRNA containing the complementarysequence (Milligan et al. (1993) J. Med. Chem. 36(14):1923-1937).Protein production is inhibited through hybridization of the antisensesequence to coding (sense) sequences in a specific mRNA target byhydrogen bonding according to Watson-Crick base pairing rules. Themechanism of antisense inhibition is that the exogenously appliedoligonucleotides decrease the mRNA and protein levels of the target gene(Milligan et al. (1993) J. Med. Chem. 36(14):1923-1937). See also Heleneand Toulme (1990) Biochim. Biophys. Acta 1049:99-125; (Cohen, J. S., ed.(1987) Oligodeoxynucleotides as antisense inhibitors of gene expression(CRC Press:Boca Raton, Fla.)).

An additional aspect of the invention includes vectors and cells forrecombinant expression of the nucleic acid molecules described herein,as well as transgenic cell populations comprising the vectors and/ornucleic acids of this invention. Also included in the invention aremethods for the expression of nucleic acids of interest of thisinvention, resulting, for example, in the production of heterologouspolypeptides and/or peptides, and methods for their use.

It is to be understood that in some embodiments of this invention, thenucleic acids of this invention encoding either a promoter or a nucleicacid of interest can be present in any number, in any order and in anycombination, either on a single nucleic acid construct or on multiplenucleic acid constructs. For example, a promoter sequence and/or anucleotide sequence of interest can be present as a single copy or asmultiple copies on the same construct and/or on multiple constructs.Also, different promoter sequences and/or different nucleotide sequencesof interest can be present on the same construct and/or on multipleconstructs in any combination of multiple and/or single copies.

Further aspects of the invention include a method of transforming a cellwith a nucleic acid and/or vector of this invention, comprisingintroducing the nucleic acid and/or vector of this invention into thecell according to methods well known in the art for transformation ofcells with nucleic acid molecules. Where the nucleic acid of interest isto be transcribed within the cell, the cell can be one in which thepromoter is operable (e.g., inducible by some stimulus such as acid pHor constitutively active). The nucleic acid of interest can be from adifferent organism than the transformed cell (e.g., a heterologousnucleic acid of interest), or the nucleic acid can be from the sameorganism as is the transformed cell, although in a recombinant nucleicacid molecule (in which case the nucleic acid of interest isheterologous in that it is not naturally occurring in the transformedcell).

A still further aspect of the invention is a method of controlling thetranscription of a nucleic acid of interest, comprising: (a) providing acell under non-inducing conditions, wherein the cell comprises arecombinant nucleic acid molecule that comprises an inducible promoterof this invention operably associated with a nucleic acid of interest;and (b) exposing, subjecting or introducing the cell to inducingconditions, e.g., an inducing environment whereby the promoter isinduced to activate transcription of the nucleic acid of interest. Theinducing environment can be an environment having a specific pH (e.g.,an acidic pH) due to an increase or decrease in the pH as compared tonon-inducing conditions, or having a specific temperature due to anincrease or decrease of temperature as compared to non-inducingconditions, or containing an inducing element (e.g., a molecule orcompound) that acts to induce the promoter to activate or increasetranscription, resulting in a level of transcription that is greaterthan the level of transcription when the inducing environment orinducing element is not present, i.e., under non-inducing conditions.Thus, a non-inducing condition is meant to include conditions whereinthe inducible promoter is not active or is not fully active in directingtranscription.

Examples of inducing elements include, but are not limited to, organicacids (lactate, acetate, oxalate), pH, sodium chloride, oxygen, hydrogenperoxide, bile, ethanol, and carbohydrates (monosaccharides,disaccharides, oligosaccharides, and galactosides such as glucose,fructose, sucrose, trehalose, fructooligosaccharide, raffinose, lactose,and galactose).

In embodiments wherein the promoter is induced by exposure to an acidicpH, the inducing step can be carried out by any suitable means,including but not limited to, adding an exogenous acid to a cell in aculture, administering or delivering a cell to an acidic body cavity ofa subject, producing an acid by fermentation in a cell culture, etc.

The nucleic acid of interest can encode various products, including butnot limited to, a protein and/or peptide (e.g., an enzyme, a hormone, agrowth factor, a cytokine, an antigen, a pro-drug, etc.) which can beboth transcribed and translated in the cell), an antisenseoligonucleotide, a ribozyme and/or an interfering RNA, etc. Suitablenucleic acids of interest can be of prokaryotic or eukaryotic origin.

As used herein, “a,” “an” or “the” can be singular or plural. Forexample, “a cell” can mean a single cell or a multiplicity of cells.

The present invention provides promoters. Thus, in some embodiments ofthe invention, a nucleic acid molecule having promoter activity isprovided comprising, consisting essentially of and/or consisting of anucleotide sequence as set forth in SEQ ID NOS:1-80 or SEQ ID NO: 6, 72,or 73 or fragments (e.g., active fragments) or active variants thereof.Also provided is a nucleic acid molecule comprising, consistingessentially of and/or consisting of a nucleic acid having promoteractivity operatively associated with a nucleic acid having activity as aregulatory element as described herein, which regulates the ability ofthe promoter sequence to activate transcription.

The nucleic acids of this invention are isolated and/or substantiallypurified. By “isolated” or “substantially purified” is meant that thenucleic acid, and/or fragments or variants, are substantially oressentially free from components normally found in association withnucleic acid in its natural state. Such components can include cellularmaterial, culture medium from recombinant production, and/or variouschemicals and reagents used in chemically synthesizing nucleic acids. An“isolated” nucleic acid of the present invention is free of nucleotidesequences that flank the nucleic acid of interest in the genomic DNA ofthe organism from which the nucleic acid was derived (such as codingsequences present at the 5′ or 3′ ends). However, the nucleic acidmolecule of this invention can, in some embodiments, include additionalbases and/or moieties that do not deleteriously affect the basiccharacteristics and/or activities of the nucleic acid. Identification ofsuch additional bases and/or moieties that do not have such adeleterious effect can be carried out by methods well known in the art.

In certain embodiments, the nucleic acid molecules of the presentinvention can be used to modulate the function of molecules. By“modulate,” “alter,” or “modify” is meant the up- or down-regulation ofa target activity. Up- or down-regulation of expression of a nucleicacid of the present invention is encompassed. Up-regulation can beaccomplished, for example, by 1) providing multiple copies of thenucleic acids of this invention, 2) modulating expression by modifyingregulatory elements, 3) promoting transcriptional or translationalmechanisms and 4) any other means known to upregulate expression ofnucleic acid. Down-regulation can be accomplished, for example, by usingwell-known antisense and gene silencing techniques. “modify” is intendedthe up- or down-regulation of a target biological activity.

By “lactic acid bacteria” is meant bacteria from a genus selected fromthe following: Aerococcus, Carnobacterium, Enterococcus, Lactococcus,Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus,Melissococcus, Alloiococcus, Dolosigranulum, Lactosphaera,Tetragenococcus, Vagococcus, and Weissella (Holzapfel et al. (2001) Am.J. Clin. Nutr. 73:365 S-373S; Williams and Wilkins (1986) Bergey'sManual of Systematic Bacteriology 2: 1075-1079 Baltimore).

By “Lactobacillus” is meant any bacteria from the genus Lactobacillus,including but not limited to L. casei, L. paracasei, L. rhamnosus, L.johnsonni, L. gasserei, L. acidophilus, L. crispatus, L. galinarum, L.plantarum, L. fermentum, L. salivarius, L. helveticus, L. bulgaricus,and numerous other species outlined by Wood et al. (Holzapfel, W. H. N.The Genera of Lactic Acid Bacteria, Vol. 2. 1995. Brian J. B. Wood, Ed.Aspen Publishers, Inc.)

The nucleic acid molecules of the present invention are also useful inmodifying milk-derived products. These uses include, but are not limitedto, modulating the growth rate of a bacterium, modifying the flavor of afermented dairy product, modulating the acidification rate of a milkproduct fermented by lactic acid bacteria, and altering productsproduced during fermentation.

In addition to the isolated nucleic acid molecules comprising nucleotidesequences as set forth in SEQ ID NOS:1-80 or SEQ ID NOS: 6, 72, or 73,the present invention also provides fragments and variants of thesenucleotide sequences. By “fragment” of a nucleotide sequence is meant anucleic acid molecule that is made up of a nucleotide sequence that isthe same as a portion of a nucleotide sequence of SEQ ID NOS:1-80, buthas fewer nucleotides than the entire nucleotide sequence as set forthin SEQ ID NOS:1-80, as well as, a nucleic acid molecule that is made upof a nucleotide sequence that has fewer nucleotides than the entirenucleotide sequence of a nucleic acid that has substantial homology to anucleotide sequence of SEQ ID NOS:1-80 as described herein and alsoincluding a nucleic acid molecule that is made up of nucleotide sequencethat has fewer nucleotides than the entire nucleotide sequence of anucleic acid that hybridizes to a nucleotide sequence of SEQ ID NOS:1-80or the complement thereof, under the conditions described herein.

In one embodiment of the invention, fragments of the polynucleotides ofSEQ ID NOS:1-80 are provided. A biologically active fragment of apolynucleotide of SEQ ID NOS:1-80 can comprise, for example, 5, 10, 15,20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or 500 contiguousnucleotides in length, including any number between 5 and 500 notspecifically recited herein, or up to the total number of nucleotidespresent in a full-length polynucleotide of the invention. Suchbiologically active fragments can continue to be biologically active(i.e., have promoter activity).

In another embodiment of the invention, fragments of the polynucleotidesof SEQ ID NOS: 6, 72 or 73 are provided. A biologically active fragmentof a polynucleotide of SEQ ID NOS:6, 72, or 73 can comprise, forexample, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or500 contiguous nucleotides in length, including any number between 5 and500 not specifically recited herein, or up to the total number ofnucleotides present in a full-length polynucleotide of the invention.Such biologically active fragments can continue to be biologicallyactive (i.e., have promoter activity).

An “active fragment” of this invention is a fragment of a nucleotidesequence of this invention that has activity, such as promoter activityand/or promoter-regulating activity as determined by any well-knownprotocol for detecting and/or measuring promoter activity and/orpromoter-regulating activity. An active fragment can also include afragment that is functional as a probe and/or primer. For example,fragments of the nucleic acids disclosed herein can be used ashybridization probes to identify nucleic acids in a sample havingvarying degrees of homology to the nucleic acid molecules of thisinvention, and/or can be used as primers in amplification protocols(e.g., polymerase chain reaction (PCR) or other well-known amplificationmethods) and/or to introduce mutations into a nucleotide sequence. Insome embodiments, fragments of this invention can be bound to a physicalsubstrate to comprise a macro- or microarray (see, for example, U.S.Pat. No. 5,837,832; U.S. Pat. No. 5,861,242; U.S. Pat. No. 6,309,823,and International Publication Nos. WO 89/10977, WO 89/11548, and WO93/17126). Such arrays of nucleic acids can be used to study geneexpression and/r to identify nucleic acid molecules with sufficientidentity to the target sequences.

A “variant” of a nucleic acid of this invention includes a nucleotidesequence that is substantially homologous to, but not identical to, anucleic acid of this invention and that retains activity as describedherein. By “substantially homologous” is meant that the variant nucleicacid has at least 50, 60, 70, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98or 99% sequence identity with a nucleic acid of this invention, asfurther described herein.

In one embodiment of the invention, variants of polynucleotides of SEQID NOS: 1-80 are provided. A variant of a polynucleotide of SEQ ID NOS:1-80 can comprise, in general, nucleotide sequences that have at leastabout 45%, 55%, 65%, 70%, 75%, 80%, 85% or 90%, 91%, 92%, 93%, 94%, 95%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NOS:1-80.Biologically active variants can continue to be biologically active(i.e., have promoter activity).

In another embodiment of the invention, variants of polynucleotides ofSEQ ID NOS:6, 72 or 73 are provided. A variant of a polynucleotide ofSEQ ID NOS:6, 72, or 73 can comprise, in general, nucleotide sequencesthat have at least about 45%, 55%, 65%, 70%, 75%, 80%, 85% or 90%, 91%,92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQID NOS:6, 72, or 73. Biologically active variants can continue to bebiologically active (i.e., have promoter activity).

The present invention further encompasses homologous nucleic acidsequences identified and/or isolated from other organisms or cells byhybridization with entire or partial nucleic acid sequences of thepresent invention, as well as, variants and/or fragments thereof. Suchhybridization protocols are standard in the art and some examples areprovided herein.

An active nucleotide fragment of this invention can be prepared byvarious methods known in the art, such as by 1) chemical synthesis, 2)restriction digestion, 3) selective amplification and 4) selectiveisolation of a desired fragment. The activity of the fragment can bedetermined by well-known methods as described herein. In someembodiments, a fragment of a nucleic acid of this invention can compriseat least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150,175, or 200 contiguous nucleotides, including any number between 5 and200 not specifically recited herein, or up to the total number ofnucleotides present in a full-length nucleotide sequence of thisinvention. The term “about”, as used herein when referring to ameasurable value such as a number of nucleotides, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

The present invention further provides nucleic acids comprisingpromoters and promoter elements that direct expression of the nucleicacids of this invention according to the methods described herein.Bacterial promoters are identified as comprising various elements thatfacilitate ribosome binding on the messenger RNA in the region upstreamof the first initiation codon of an open reading frame. These elementscan include a hexamer region centered around nucleotide −10 and/oranother hexamer centered around nucleotide −35, counting upstream fromthe first nucleotide of the initiation codon in negative numbers. Anexample of a −10 hexamer is TATAAT (SEQ ID NO: 109) and an example of a−35 hexamer is TTGACA ((SEQ ID NO: 110) e.g., as part of TCTTGACAT) (SEQID NO: 111). These hexamers are recognized by the σ subunit of the RNApolymerase. There is also a spacer region connecting these two hexamers,the length of which is commonly conserved in most bacteria to be 17±5base pairs. A TG motif upstream of the −10 hexamer is also commonlyfound in bacterial promoters, as well as an UP element, which is anAT-rich sequence upstream of the −35 hexamer (e.g., commonly around −40to −60). This latter element is contacted by the C-terminal domain ofthe RNA polymerase α-subunit. Nonlimiting examples of a consensussequence for an UP element of a bacterial promoter of this inventioninclude

(SEQ ID NO:81) nnAAA(A/T)(A/T)T(A/T)TTTTnnAAAAnnn, (SEQ ID NO:82)NNAWWWWWTTTTTN, (SEQ ID NO:83) AAAAAARNR, (SEQ ID NO:84)NNAAAWWTWTTTTNNNAAANNN, (SEQ ID NO:85) AAAWWWTWTTTTNNNAAA and (SEQ IDNO:86) GNAAAAATWTNTTNAAAAAAMNCTTGMA(N)₁₈TATAAT,where W is A or T; M is A or C; R is A or G; and N is any base. Alsoincluded are complements of these sequences.

Thus, in certain embodiments, the present invention provides an isolatednucleic acid comprising from about 50 or 75 to about 100, 125, 150, 175,or 200 contiguous nucleotides, including any number between 50 and 200not specifically recited herein (e.g., 60, 75, 96, 179, etc.), saidnucleotides being located immediately upstream of the initiation codonof an open reading frame of this invention or upstream of a sequencecorresponding to tRNA or rRNA, and numbered from between −1 to −200 inthe nucleotide sequence, starting with the first nucleotide of theinitiation codon or tRNA or rRNA sequence and numbering backwards innegative numbers, and further wherein said sequence of contiguousnucleotides comprises one or more of the promoter elements describedherein and/or one or more nucleotide sequences having substantialsimilarity to a promoter element described herein and wherein saidnucleic acid has promoter activity and/or potential promoter activity asdetected according to methods standard in the art. (See, e.g., McCrackenet al. (2000) “Analysis of promoter sequences from Lactobacillus andLactococcus and their activity in several Lactobacillus species” Arch.Microbiol. 173:383-389; Estrem et al. (1998) “Identification of an UPelement consensus sequence for bacterial promoters” Proc. Natl. Acad.Sci. USA 95:9761-9766; Ross et al. (1998) “Escherichia coli promoterswith UP elements of different strengths: Modular structure of bacterialpromoters” J. Bacteriol. 180:5375-5383; U.S. Pat. No. 6,605,431 toGourse et al., entitled “Promoter elements and methods of use”; and PCTpublication number WO 2004/067772, published Aug. 12, 2004 and entitled“Method for the identification and isolation of strong bacterialpromoters.”) Each of these references is incorporated herein in itsentirety for teachings regarding bacterial promoters and bacterialpromoter elements and for additional examples of nucleotide sequences ofbacterial promoter elements described herein.

The nucleic acid molecules of this invention comprising promoters and/orpromoter elements have practical utility in the regulation of expressionof homologous and/or heterologous nucleic acids, for example, to produceproteins for use in the various embodiments of this invention, as wellas in any commercial application, such as, e.g., bacterial fermentationprocesses (e.g., production of insulin, blood coagulation proteins,etc.)

In some embodiments, the nucleic acid molecules of this inventioncomprise a regulatory element that modulates the ability of the promoterto activate transcription. Regulatory elements of the present inventionare generally located within the approximately 0.2 kb of DNA 5′ to theopen reading frames of the Lactobacillus acidophilus NCFM genome. Itwill be apparent that other sequence fragments, longer or shorter thanthe foregoing sequence, or with minor additions, deletions, orsubstitutions made thereto, as those that result, for example fromsite-directed mutagenesis, as well as from synthetically derivedsequences, are included within the present invention.

In one embodiment of the invention, a nucleic acid molecule of thisinvention comprises a regulatory element that is a catabolite responseelement (cre). By “catabolite response element,” “cre sequence” or“cre-like sequence” is meant a cis-acting DNA sequence involved incatabolite repression. Expression of many catabolic enzymes ingram-positive bacteria is subject to repression by glucose and otherrapidly metabolizable sources of carbon (Stewart (1993) J. Cell.Biochem. 51:25-28; Hueck and Hillen (1995) Mol. Microbiol. 143:147-148).This catabolite repression of such genes in gram-positive bacteria,notably Bacillus subtilis, is under the control of cis-acting nucleotidesequences described as cre sequences. These sequences contain a 2-foldaxis of symmetry, are generally located in the region of promoterelements, can be present in multiples (e.g., pairs), and can vary insequence location relative to the transcription start site for thetranscription product under control of a given promoter element.Consensus nucleotide sequences for cre sequences are known in the art.Nonlimiting examples of consensus cre sequences include TGWAANCGNTNWCA(SEQ ID NO:87) (Weickert and Chambliss. 1990 Proc. Natl. Acad. Sci. USA87:6238-6242); WWWWTGWAARCGYTWNCWWWW (SEQ ID NO:88)(Zallieckas et al.(1998) J. Bacteriol. 180:6649-6654); and WWTGNAARCGNWWWCAWW (SEQ IDNO:89) (Miwa et al. (2000) Nucleic Acids Res. 28:1206-1210). Thus, insome embodiments, the present invention provides promoter sequencescomprising one or more cre sequences. In certain embodiments, a promotersequence of this invention can comprises one, two, or more than two cresequences. Furthermore, the present invention provides fragments of thepromoters of this invention, wherein the fragment comprises and/orconsists essentially of a consensus cre sequence and/or a sequence thatcan be up to 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to aconsensus cre sequence.

The regulatory elements of this invention that enhance activation oftranscription can increase nucleic acid transcription by at least 50%,60%, 70%, 80%, 90%, 100%, 150%, 200%, 300% or more. The regulatoryelements of this invention that suppress transcription can do so by atleast 25%, 35%, 50%, 60%, 75%, 85%, 95% or more, up to and including100%.

In other embodiments, the sequence of the nucleic acid encoding theregulatory element can correspond to a portion of the nucleotidesequence of a nucleic acid of this invention, such as the nucleotidesequences as set forth in SEQ ID NOS:1-80 or SEQ ID NO: 6, 72, or 73.Also included herein are fragments of a nucleotide sequence that is aregulatory element, wherein the fragment retains activity of theregulatory element. Nucleic acids of this invention that are fragmentsof a promoter or regulatory element can comprise, consist essentially ofand/or consist of at least 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125,150, 175, or 200 contiguous nucleotides of the full-length sequence.Particular fragment lengths will depend upon the objective and will alsovary depending upon the particular promoter or regulatory sequence.

The nucleotides of such fragments will usually comprise the TATArecognition sequence of the particular promoter sequence. Such fragmentscan be obtained by use of restriction enzymes to cleave the naturallyoccurring promoter nucleotide sequence disclosed herein; by synthesizinga nucleotide sequence from the naturally occurring sequence of thepromoter nucleic acid sequence; or through the use of amplificationprotocols, such as PCR. See, for example, Mullis et al. (1987) MethodsEnzymol. 155:335-350, and Erlich, ed. (1989) PCR Technology (StocktonPress, New York). Variants of these promoter fragments, such as thoseresulting from site-directed mutagenesis, are also encompassed in thepresent invention, as such variants are described herein.

Regulatory elements of the present invention can also include nucleicacids that regulate expression of nucleic acids and have a sequence thatis substantially homologous to a nucleotide sequence comprising aregulatory element as disclosed herein, and particularly a nucleotidesequence comprising a regulatory element as disclosed herein as SEQ IDNOS:1-80.

Thus, a nucleic acid encoding a regulatory element of this inventionincludes a nucleic acid that is at least about 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous to a nucleic acidencoding a regulatory element as described herein, and in particular anucleic acid encoding a regulatory element and having the nucleotidesequence set forth herein as SEQ ID NOS:1-80 or SEQ ID NOS: 6, 72, or73. Regulatory elements from other species are also encompassed hereinand include those that are at least about 75%, 80%, 85%, 90% or 95%homologous to a continuous segment of a regulatory element of thepresent invention, and which are capable of regulating the activation oftranscription of nucleic acids.

As used herein, two nucleotide sequences are “substantially homologous”when they have at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% homology with one another.

The term “homology” as used herein refers to a degree of similaritybetween two or more sequences. There can be partial homology or completehomology (i.e., identity). A partially homologous nucleic acid sequencethat at least partially inhibits a complementary nucleic acid sequencefrom hybridizing to a target nucleic acid is referred to using thefunctional term “substantially homologous.” The inhibition ofhybridization to the target sequence can be examined using ahybridization assay (Southern or Northern blot, solution hybridizationand the like) under conditions of varying stringency, as that term isknown in the art. A substantially homologous sequence or hybridizationprobe will compete for and inhibit the binding of a completelycomplementary sequence to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding canbe tested by the use of a second target sequence, which lacks even apartial degree of complementarity (e.g., less than about 30%). In theabsence of non-specific binding, the probe will not hybridize to thesecond non-complementary target sequence.

Alternatively stated, in particular embodiments, nucleic acids thathybridize under the conditions described herein to the complement of thesequences specifically disclosed herein can also be used according tothe present invention. The term “hybridization” as used herein refers toany process by which a first strand of nucleic acid binds with a secondstrand of nucleic acid through base pairing.

The term “stringent” as used here refers to hybridization conditionsthat are commonly understood in the art to define the conditions of thehybridization procedure. Stringency conditions can be low, high ormedium, as those terms are commonly know in the art and well recognizedby one of ordinary skill. High stringency hybridization conditions thatwill permit a complementary nucleotide sequence to hybridize to anucleotide sequence as given herein are well known in the art. As oneexample, hybridization of such sequences to the nucleic acid moleculesdisclosed herein can be carried out in 25% formamide, 5×SSC,5×Denhardt's solution and 5% dextran sulfate at 42° C., with washconditions of 25% formamide, 5×SSC and 0.1% SDS at 42° C., to allowhybridization of sequences of about 60% homology. Another exampleincludes hybridization conditions of 6×SSC, 0.1% SDS at about 45° C.,followed by wash conditions of 0.2×SSC, 0.1% SDS at 50-65° C. Anotherexample of stringent conditions is represented by a wash stringency of0.3M NaCl, 0.03M sodium citrate and 0.1% SDS at 60-70° C. using astandard hybridization assay (see Sambrook et al., eds., MolecularCloning: A Laboratory Manual 2d ed. (Cold Spring Harbor, N.Y. 1989, theentire contents of which are incorporated by reference herein). Invarious embodiments, stringent conditions can include, for example,highly stringent (i.e., high stringency) conditions (e.g., hybridizationto filter-bound DNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS) and1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C.), and/ormoderately stringent (i.e., medium stringency) conditions (e.g., washingin 0.2×SSC/0.1% SDS at 42° C.).

As is known in the art, a number of different programs can be used toidentify whether a nucleic acid or amino acid has homology (e.g.,sequence identity or similarity) to a known sequence. Homology can bedetermined using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman (1981) Adv. Appl. Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, thesearch for similarity method of Pearson and Lipman (1988) Proc. Natl.Acad. Sci. USA 85:2444, computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Drive, Madison, Wis.)and/or the Best Fit sequence program described by Devereux et al. (1984)Nucl. Acid Res. 12:387-395, preferably using the default settings, or byinspection.

An example of a useful algorithm is PILEUP, which creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng and Doolittle (1987) J. Mol.Evol. 35:351-360; which is similar to that described by Higgins andSharp (1989) CABIOS 5:151-153.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., Proc. Natl. Acad. Sci. USA 90, 5873-5787 (1993). A particularlyuseful BLAST program is the WU-BLAST-2 program that was obtained fromAltschul et al., Methods in Enzymology, 266, 460-480 (1996). WU-BLAST-2uses several search parameters, which are preferably set to the defaultvalues. The parameters are dynamic values and are established by theprogram itself depending upon the composition of the particular sequenceand composition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity. An additional useful algorithm is gapped BLAST asreported by Altschul et al. Nucleic Acids Res. 25, 3389-3402.

The CLUSTAL program can also be used to determine sequence similarity.This algorithm is described by Higgins et al. (1988) Gene 73:237;Higgins et al. (1989) CABIOS 5:151-153; Corpet et al. (1988) NucleicAcids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; andPearson et al. (1994) Meth. Mol. Biol. 24: 307-331.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix or any equivalent program thereof. Otherequivalent programs can also be used. By “equivalent program” is meantany sequence comparison program that, for any two sequences in question,generates an alignment having identical nucleotide or amino acid residuematches and an identical percent sequence identity when compared to thecorresponding alignment generated by GAP Version 10.

In addition, for sequences that contain either more or fewer nucleotidesthan the nucleic acids disclosed herein, it is understood that in oneembodiment, the percentage of sequence homology will be determined basedon the number of identical nucleotides in relation to the total numberof nucleotide bases. Thus, for example, sequence homology of sequencesshorter than a sequence specifically disclosed herein can be determinedusing the number of nucleotide bases in the shorter sequence, in oneembodiment. In percent homology calculations, relative weight is notassigned to various manifestations of sequence variation, such as,insertions, deletions, substitutions, etc.

The present invention also provides a recombinant nucleic acidcomprising a nucleic acid encoding a regulatory element operablyassociated with a nucleic acid of interest. The nucleic acid encodingthe regulatory element is operably associated with the nucleic acid ofinterest such that the regulatory element can modulate transcription ofthe nucleic acid of interest as directed by a nucleic acid havingpromoter activity. Typically, the nucleic acid encoding the regulatoryelement and/or the nucleic acid having promoter activity will be located5′ to the nucleic acid of interest, but either or both can also belocated 3′ to the nucleic acid of interest as long as they are operablyassociated therewith. There are no particular upper or lower limits asto the distance between the nucleic acid encoding the regulatory elementand/or the nucleic acid having promoter activity and the nucleic acid ofinterest, as long as the nucleic acids are operably associated with oneanother.

The nucleic acid molecules of the present invention can also be includedin vectors, which in some embodiments can be expression vectors. Avector of this invention can include one or more regulatory sequences todirect the expression of nucleic acids to which they are operably linkedor operatively associated. The term “regulatory sequence” is meant toinclude, but is not limited to, promoters, operators, enhancers,transcriptional terminators, and/or other expression control elementssuch as translational control sequences (e.g., Shine-Dalgarno consensussequence, initiation and termination codons). These regulatory sequenceswill differ, for example, depending on the cell into which the vector isto be introduced.

The vectors of this invention can be autonomously replicated in a cell(episomal vectors), or they can be integrated into the genome of a cell,and replicated along with the cell's genome (non-episomal vectors).Integrating vectors in prokaryotes typically contain at least onesequence homologous to the bacterial chromosome that allows forrecombination to occur between homologous nucleic acid in the vector andthe bacterial chromosome. Integrating vectors can also comprisebacteriophage or transposon sequences. Episomal vectors, or plasmids aretypically circular double-stranded nucleic acid loops into whichadditional nucleic acid sequences can be ligated.

The vectors of this invention can comprise a nucleic acid of thisinvention in a form suitable for expression of the nucleic acid in acell, which can be a eukaryotic or prokaryotic cell. It will beappreciated by those skilled in the art that the design of the vectorcan depend on such factors as the choice of the cell to be transformed,the level of expression of nucleic acid and/or production of proteindesired, etc.

A promoter of this invention can be regulated in its transcriptionactivity in various ways, as are known to one of ordinary skill in theart. For example, regulation can be achieved in some embodiments when agene activator protein sequence is present. When present, such asequence is usually proximal (5′) to the RNA polymerase bindingsequence.

An example of a gene activator protein is the catabolite activatorprotein (CAP), which helps initiate transcription of the lac operon inEscherichia coli (Raibaud et al. (1984) Annu. Rev. Genet. 18:173).Regulated expression can therefore be either positive or negative,thereby either enhancing or reducing transcription. Other examples ofpositive and negative regulatory elements are well known in the art.Various other promoters besides the promoters of this invention can beincluded in the vectors of this invention. Examples of such otherpromoters include, but are not limited to, a T7/LacO hybrid promoter, atrp promoter, a T7 promoter, a lac promoter, and a bacteriophage lambdapromoter. Such other promoters can be constitutively active orinducible.

It is also contemplated that the promoters of the present invention canbe combined with synthetic promoters that do not occur in nature, and/orsuch synthetic promoters can be present in a vector of this invention,in combination with a promoter of this invention. For example,transcription activation sequences of one bacterial or bacteriophagepromoter may be joined with the operon sequences of another bacterial orbacteriophage promoter, creating a synthetic hybrid promoter (U.S. Pat.No. 4,551,433). For example, the tac (Amann et al. (1983) Gene 25:167;de Boer et al. (1983) Proc. Natl. Acad. Sci. 80:21) and trc (Brosius etal. (1985) J. Biol. Chem. 260:3539-3541) promoters are hybrid trp-lacpromoters comprised of both trp promoter and lac operon sequences thatare regulated by the lac repressor. The tac promoter has the additionalfeature of being an inducible regulatory sequence. Thus, for example,expression of a coding sequence operably linked to the tac promoter canbe induced in a cell culture by adding isopropyl-1-thio-β-D-galactoside(IPTG).

Furthermore, a vector of this invention can include naturally occurringpromoters of non-bacterial origin that have the ability to bindbacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somenucleic acids in prokaryotes. The bacteriophage T7 RNApolymerase/promoter system is an example of a coupled promoter system(Studier et al. (1986) J. Mol. Biol. 189:113; Tabor et al. (1985) Proc.Natl. Acad. Sci. 82:1074). In addition, a hybrid promoter is alsoprovided, which can comprise a bacteriophage promoter and a promoter oractive region of a promoter of the present invention.

The vector of this invention can additionally comprise a nucleic acidsequence encoding a repressor (or inducer) for the promoter provided inthe vector. For example, an inducible vector of the present inventionmay regulate transcription from the Lac operator (LacO) by expressingthe gene encoding the LacI repressor protein. Other examples include theuse of the lexA gene to regulate expression of pRecA, and the use oftrpO to regulate ptrp. Alleles of such genes that increase the extent ofrepression (e.g., lacIq) or that modify the manner of induction (e.g.,λCI857, rendering λpL thermo-inducible, or λCI+, rendering λpLchemo-inducible) may be employed.

In addition to a functioning promoter sequence, an efficientribosome-binding site is also useful for the expression of nucleic acidsequences from the vectors of this invention. In prokaryotes, theribosome binding site is called the Shine-Dalgarno (SD) sequence andincludes an initiation codon (ATG) and a sequence 3-9 nucleotides inlength located 3-11 nucleotides upstream of the initiation codon (Shineet al. (1975) Nature 254:34). The SD sequence is thought to promotebinding of mRNA to the ribosome by the pairing of bases between the SDsequence and the 3′ end of bacterial 16S rRNA (Steitz et al. (1979)“Genetic Signals and Nucleotide Sequences in Messenger RNA,” inBiological Regulation and Development: Gene Expression (ed. R. F.Goldberger, Plenum Press, NY).

The nucleic acid of interest provided in this invention can encode apeptide and/or polypeptides that can be secreted from the cell. Such apeptide or polypeptide is produced by creating chimeric nucleic acidmolecules that encode a protein or peptide comprising a signal peptidesequence that provides for secretion of polypeptides in bacteria (U.S.Pat. No. 4,336,336). The signal sequence typically encodes a signalpeptide comprised of hydrophobic amino acids that direct the secretionof the protein or peptide from the cell. The protein or peptide iseither secreted/exported into the growth medium (gram-positive bacteria)or into the periplasmic space, located between the inner and outermembrane of the cell (gram-negative bacteria). In some embodiments,processing sites can be introduced, where cleavage can occur, either invivo or in vitro, located between the signal peptide sequence and thepeptide or polypeptide.

Nucleic acids encoding suitable signal sequences can be derived fromgenes encoding secreted bacterial proteins, such as the E. coli outermembrane protein gene (ompA) (Masui et al. (1983) FEBS Lett.151(1):159-164; Ghrayeb et al. (1984) EMBO J. 3:2437-2442) and the E.coli alkaline phosphatase signal sequence (phoA) (Oka et al. (1985)Proc. Natl. Acad. Sci. 82:7212). Other prokaryotic signal sequences caninclude, for example, the signal sequence from penicillinase, Ipp, orheat stable enterotoxin II leaders.

The vectors of this invention can further comprise a transcriptiontermination sequence. Typically, transcription termination sequencesrecognized by bacteria are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter, flank thecoding sequence of a nucleic acid of interest. These sequences directthe transcription of a mRNA that can be translated into the polypeptideor peptide or other gene product encoded by the nucleic acid ofinterest. Transcription termination sequences frequently include nucleicacid sequences (of about 50 nucleotides) that are capable of formingstem loop structures that aid in terminating transcription. Examplesinclude transcription termination sequences derived from genes withstrong promoters, such as the trp gene in E. coli as well as otherbiosynthetic genes.

The vectors of this invention can also comprise at least one, andtypically a plurality of restriction sites for insertion of the nucleicacid(s) of interest so that it is under transcriptional regulation ofthe regulatory regions. Selectable marker genes that ensure maintenanceof the vector in the cell can also be included in the vector. Examplesof selectable markers include, but are not limited to, those that conferresistance to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin (neomycin), and tetracycline (Davies et al. (1978) Annu. Rev.Microbiol. 32:469). Selectable markers can also allow a cell to grow onminimal medium, or in the presence of toxic metabolites and can includebiosynthetic genes, such as those in the histidine, tryptophan, andleucine biosynthetic pathways.

Regulatory regions present in the vector of this invention can be native(homologous), or foreign (heterologous) to the host cell and/or to thepromoter and/or nucleic acid of interest of this invention. Theregulatory regions can also be natural or synthetic. By “operablylinked” is meant that the nucleotide sequence of interest is linked tothe regulatory sequence(s) such that expression of the nucleotidesequence is allowed (e.g., in an in vitro transcription/translationsystem or in a cell when the vector is introduced into the cell). Asused herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide. Inanother example, where the region is “foreign” or “heterologous” to thehost cell, it can mean that the region is not found in the native cellinto which the region is introduced. Alternatively, where the region is“foreign” or “heterologous” to the promoter and/or nucleic acid ofinterest of the invention, it is meant that the region is not the nativeor naturally occurring region for the operably linked promoter and/ornucleic acid of interest of the invention. For example, the regulatoryregion can be derived from phage. While it may be preferable to expressthe nucleic acid of interest using heterologous regulatory regions,native regions can also be used. Such constructs would be expected insome cases to alter expression levels of nucleic acids in the host cell.Thus, the phenotype of the host cell could be altered.

In preparing the vector of this invention, the various nucleotidesequences can be manipulated, so as to position the promoter and/ornucleic acid of interest and/or other regulatory elements in the properorientation in the vector and, as appropriate, in the proper readingframe. Toward this end, adapters or linkers can be employed to join thenucleotide sequences or other manipulations can be employed to providefor convenient restriction sites, removal of superfluous nucleic acid,removal or addition of restriction sites, and the like as would be wellknown in the art. For this purpose, in vitro mutagenesis, primer repair,restriction, annealing, resubstitutions, e.g., transitions andtransversions, can be employed, according to art-known protocols.

The invention further provides a vector comprising a nucleic acidmolecule of this invention cloned into the vector in an antisenseorientation. That is, the nucleic acid is operably linked to a promoterof this invention in a manner that allows for expression (bytranscription of the nucleic acid molecule) of an RNA molecule that isantisense to a messenger RNA in a cell into which the vector isintroduced. The promoter operably linked to the nucleic acid cloned inthe antisense orientation can be chosen to direct the continuous orinducible expression of the antisense RNA molecule. In some embodiments,the antisense vector can be in the form of a recombinant plasmid orphagemid in which antisense nucleic acids are produced under the controlof a high efficiency regulatory region comprising a promoter of thisinvention, the activity of which can be determined by the cell type intowhich the vector is introduced. For a discussion of the regulation ofprotein production in a cell using antisense sequences, see Weintraub etal. (1986) Reviews—Trends in Genetics, Vol. 1 (1).

In some embodiments of the present invention, the production of bacteriacontaining the recombinant nucleic acid sequences of this invention, thepreparation of starter cultures of such bacteria, and methods offermenting substrates, particularly food substrates such as milk, can becarried out in accordance with known techniques. (See, for example,Gilliland, S. E. (ed) Bacterial Starter Cultures for Food, CRC Press,1985, 205 pp.; Read, G. (Ed.). Prescott and Dunn's IndustrialMicrobiology, 4^(th) Ed. AVI Publishing Company, Inc. 1982, 883 pp.;Peppler, J. J. and Perlman, D. (Eds.). Microbiol Technology: Volume II,Fermentation Technology, Academic Press, 1979, 536 pp.)

By “fermenting” is meant the energy-yielding, metabolic breakdown oforganic compounds by microorganisms that generally proceeds underanaerobic conditions and with the production of organic acids (lactate,acetate) as major end products and minor end products, such as ethanol,carbon dioxide and diacetyl.

By “introducing” as it pertains to nucleic acid molecules is meantintroduction into cells, such as prokaryotic cells via conventionaltransformation or transfection techniques, or by phage-mediatedinfection. As used herein, the terms “transformation,” “transduction,”“conjugation,” and “protoplast fusion” are meant to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a eukaryotic or prokaryotic host cell, including calciumphosphate or calcium chloride co-precipitation, DEAE-dextran-mediatedtransfection, lipofection, or electroporation. Suitable methods fortransforming or transfecting host cells can be found in Sambrook et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold SpringHarbor Laboratory Press, Plainview, N.Y.) and other laboratory manuals.By “introducing” or “delivering” as it pertains to cells such asbacterial cells of the invention, is meant introduction into a subjectby ingestion, topical application, nasal, urogenital, suppository,and/or oral application of the microorganism.

Bacterial cells of this invention are cultured in suitable medium, asdescribed generally in Sambrook et al. (1989) Molecular Cloning, ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.). The nucleic acids and/or vectors of this invention canbe used to transform cells, which can be in vitro or in vivo. Thus, thepresent invention further provides a method of transforming a cell,comprising introducing a nucleic acid and/or vector of this inventioninto the cell according to well-known methods for transforming cells, asdescribed herein. The cell of this invention can be a prokaryotic cellor a eukaryotic cell.

As indicated herein, in some embodiments, the nucleic acid and/or vectorof this invention can be introduced into a bacterial cell and thebacterial cell can be administered to a subject that can safely receivethe bacterial cell.

In embodiments wherein the cell of this invention is in vivo, thenucleic acid and/or vector of this invention can be delivered to orintroduced into a subject comprising the cell.

A subject of this invention can be any animal having cells that can betransformed by the nucleic acids and/or vectors of this invention and/orhaving the capability of receiving transformed bacterial cells of thisinvention. The animal can be a mammal, an avian species, a reptile, orany other type of animal. In some embodiments, the animal is a mammal,which can be a domesticated animal (e.g., cat, dog, horse, cow, goat), ahuman or a non-human primate.

Experimental

Microarray Construction. A whole genome DNA microarray based on the PCRproducts of predicted ORFs from the L. acidophilus genome was used forglobal gene expression analysis. PCR primers for 1,966 genes weredesigned using GAMOLA software (Altermann and Klaenhammer 2003 “GAMOLA:a new local solution for sequence annotation and analyzing draft andfinished prokaryotic genomes” OMICS 7:161-169) and purchased from QiagenOperon (Alameda, Calif.). Total genomic DNA from L. acidophilus NCFM wasused as a template for 96-well PCR amplifications. To amplifygene-specific PCR products, a 100 μl reaction mix contained: 1 μl L.acidophilus DNA (100 ng/ml), 10 μl specific primer pairs (10 μM), 0.5 μlof dNTP mix (10 mM), 10 μl PCR buffer (10×), and 1 μl Taq DNA polymerase(5 U/μl [Roche Molecular Biochemicals]). The following PCR protocol wasused: an initial denaturation step for 5 min at 94° C. followed by 40cycles of denaturation at 94° C. for 15 sec, annealing at 50° C. for 30sec and polymerization at 72° C. for 45 sec. Approximately 95% of openreading frames (ORFs) produced a unique PCR product between 100-800 bp.The size of fragments was confirmed by electrophoresis in 1% agarosegels. DNA from 96-well plates was purified using the Qiagen PurificationKit. In general, the total quantity of each PCR product was greater than1 μg.

The purified PCR fragments were spotted three times in a random patternon glass slides (Corning, Acton, Mass.) using the Affymetrix® 417™Arrayer at the NCSU Genome Research Laboratory. To prevent carry-overcontaminations, pins were washed between uses in different wells.Humidity was controlled at 50-55% during printing. DNA was cross-linkedto the surface of the slide by UV (300 mJ) and posterior incubation ofthe slides for 2 h at 80° C. The reliability of the microarray data wasassessed by hybridization of two cDNA samples prepared from the sametotal RNA, labeled with Cy3 and Cy5. Hybridization data revealed alinear correlation in the relative expression level of 98.6% of 5685spots (each gene by triplicate) with no more than a two-fold change.

Culture treatment/growth conditions. The strain used in this study is L.acidophilus NCFM (NCK56) (Altermann et al. 2004 “Identification andphenotypic characterization of the cell-division protein CdpA” Gene342:189-197). For the studies examining growth on varying carbohydrates,cultures were propagated at 37° C., aerobically in MRS broth (Difco). Asemi-synthetic medium consisted of: 1% bactopeptone (w/v) (Difco), 0.5%yeast extract (w/v) (Difco), 0.2% dipotassium phosphate (w/v) (Fisher),0.5% sodium acetate (w/v) (Fisher), 0.2% ammonium citrate (w/v) (Sigma),0.02% magnesium sulfate (w/v) (Fisher), 0.005% manganese sulfate (w/v)(Fisher), 0.1% Tween 80 (v/v) (Sigma), 0.003% bromocresol purple (v/v)(Fisher) and 1% sugar (w/v). The carbohydrates added were either:glucose (dextrose) (Sigma), fructose (Sigma), sucrose (Sigma), FOS(raftilose P95) (Orafti), raffinose (Sigma), lactose (Fisher), galactose(Sigma) or trehalose (Sigma). Without carbohydrate supplementation, thesemi-synthetic medium was unable to sustain bacterial growth. Cellsunderwent at least five passages on each sugar prior to RNA isolation,to minimize carryover between substrates (Chhabra et al.“Carbohydrate-induced differential gene expression patterns in thehyperthermophilic bacterium Thermotoga maritima.” J Biol Chem. (2003)278(9):7540-52). In the final culture, L. acidophilus cells wereinoculated into semi-synthetic medium supplemented with 1% (w/v) selectsugars and propagated to mid-log phase (OD_(600 nm)˜0.6). Cells wereharvested by centrifugation (2 minutes, 14,000 rpm) and immediatelycooled on ice prior to RNA isolation.

For studies on cells exposed to varying stresses, L. acidophilus NCFMwas grown from a 2% inoculum in MRS broth to OD₆₀₀ of 0.25-0.3 (pH>5.8).Cultures were centrifuged and resuspended in the same volume of MRSadjusted to pH 5.5 or 4.5 with lactate, MRS containing 5% bile, 70 mMammonium oxalate or 15% ethanol (v/v) and incubated at 37° C. for 30min. After incubation, cells were harvested by centrifugation and frozenimmediately in a dry ice/ethanol bath.

Measurement of GUS activity. L. acidophilus cultures were grown tomid-log phase (OD=0.5) in MRS, harvested and resuspended in SSM+1%carbohydrate, incubated at 37° C. for up to three hours and then thecells were harvested by centrifugation. Cell pellets were resuspended in1 mL GUS assay buffer (100 mM sodium phosphate, 2.5 mM EDTA, pH 6.0) andtransferred to tubes containing glass beads for bead beating (3×1 minwith 1 min rest on ice between cycles). Cell debris was pelleted andprotein concentration was determined via the Bradford method. GUSactivity for 1 mg of protein was then determined spectrophometricallyusing MUG as the substrate under the following conditions: 100 mMNa-phosphate, 2.5 mM EDTA 1 mM MUG, pH 6.0 at 37° C. Fluorescence wasmeasured using a Fluostar Optima microplate reader with excitation at355 nm and emission at 460 nm. A standard curve for4-methylumbelliferone (10 to 600 nM) in GUS lysis buffer also wasgenerated, and GUS activity was expressed in pmol 4-methylumbelliferoneproduced per minute per milligram of protein. Such methods were carriedout to obtain the data appearing in Table 1 and FIGS. 2 and 3.

RNA isolation. Total RNA was isolated using TRIzol (GibcoBRL) byfollowing the manufacturer's instructions. Pellets were resuspended inTRIZOL, by vortexing and underwent five cycles of 1 min bead beating and1 min on ice. Nucleic acids were purified using three chloroform(Fisher) extractions, and precipitated using isopropanol (Fisher) andcentrifugation for 10 min at 12,000 rpm. The RNA pellet was washed with70% ethanol (AAPER Alcohol and Chemical co.) and resuspended intoDEPC-(Sigma) treated water. RNA samples were treated with DNAse Iaccording to the manufacturer's recommendations (Boehringer Mannheim).

cDNA target preparation and microarray hybridization. For eachhybridization, RNA samples (25 μg of DNase treated) were amino-allyllabeled by reverse transcription using random hexamers (Invitrogen LifeTechnologies, Carlsbad, Calif.) as primers, in the presence ofamino-allyl dUTP (Sigma, Town, state), by a SuperScript II reversetranscriptase (Invitrogen Life Technologies, Carlsbad, Calif.), asdescribed previously (Hedge et al. 2000 “A concise guide to cDNAmicroarray analysis” Biotechniques 29(3):548-50; Azcarate-Peril et al.2004 “Identification and inactivation of genetic loci involved withLactobacillus acidophilus acid tolerance” Appl. Environ. Microbiol.70:5315-5322). Labeled cDNA samples were subsequently coupled witheither Cy3 or Cy5 N-hydroxysuccinimidyl-dyes (Amersham BiosciencesCorp., Piscataway, N.J.), and purified using a PCR purification kit(Qiagen). The resulting samples were hybridized onto microarray slidesand further processed as described previously (Azcarate-Peril et al.2004), according to the TIGR protocol (Hedge et al. 2000). Briefly,combined Cy5- and Cy3-labeled cDNA probes were hybridized to the arraysfor 16 h at 42° C. After hybridization, the slides were washed twice inlow stringency buffer (1×SSC containing 0.2% SDS) for 5 min each. Thefirst wash was performed at 42° C. and the second one at roomtemperature. Subsequently, the slides were washed in a high stringencybuffer (0.1×SSC containing 0.2% SDS, for 5 min at room temperature) andfinally in 0.1×SSC (2 washes of 2.5 min each at room temperature).

For stress microarray hybridizations a Reference Sample design was used,where each sample was compared using a dye swap to a common referencesample (early log-phase L. acidophilus cultures resuspended in fresh MRS[pH ˜6.8]), so that experiments could be extended to assay severalsamples collected over a period of time, all comparisons were made withequal efficiency and every new sample in a reference experiment wasmanaged in the same way.

Hybridizations in sugar experiments were performed according to a singleRound-Robin design, so that all possible direct pair-wise comparisonswere conducted (See Figure below). With 8 different sugars, a total of28 hybridizations were performed. Each treatment was labeled 7 times,and every-other treatment was labeled with either Cy3 or Cy5, 4 and 3times, alternatively.

Microarray data collection and analysis. Microarray images were acquiredusing a Scanarray 4000 Microarray Scanner (Packard Biochip Bioscience,MA). Signal fluorescence, including spot and background intensities weresubsequently quantified and assigned to genomic ORFs using Quantarray3.0 (Packard BioChip Technologies LLC, Billerica, Mass.).

Data normalization and gene expression analysis. Immediately afterwashing of the arrays, fluorescence intensities were acquired at 10 μmresolution using a ScanArray 4000 Microarray Scanner (Packard BiochipBioScience, Biochip Technologies LLC, Mass.) and stored as TIFF images.Signal intensities were quantified, the background was subtracted anddata were normalized using the QuantArray 3.0 software package (PerkinElmer). Two slides (each containing triplicate arrays) were hybridizedreciprocally to Cy3- and Cy5-labeled probes per experiment (dye swap).Spots were analyzed by adaptive quantitation. Data were mediannormalized. When the local background intensity was higher than the spotsignal (negative values) no data were considered for those spots. Themedian of the six ratios per gene was recorded. The ratio between theaverage absolute pixel values for the replicated spots of each gene withand without treatment represented the fold change in gene expression.All genes belonging to a potential operon were considered for analysisif at least one gene of the operon showed significant expression changesand the remaining genes showed trends toward that expression. Confidenceintervals and P values on the fold change were also calculated with theuse of a two-sample t test. P values of 0.05 or less were consideredsignificant.

Table 3 provides the nucleotide sequence of promoters as identified fromthe sequencing and characterization of the genome of Lactobacillusacidophilus NCFM. The sequences are shown to be identified by the openreading frame with which the promoter sequence is associated in thegenome and the expression conditions studied, with results described. Apredicted ribosome binding site (RBS) is underlined for each promotersequence in the figure. The expression characteristics of various genesand their corresponding ORF# under control of the promoters of thisinvention are shown under each sequence appearing in Table 3. Genes thatare “consistently highly”, the genes expressed by promoters responsiveto the “stress” conditions described herein, and the genes that areexpressed under the control of the promoters responsive to the sugarsdescribed herein are summarized in Table 3.

Table 4 provides a listing of all promoters shown in Table 3 that havecre elements as described herein, as well as a summary of the number ofpromoters described in Table 3 and the number of genes that areexpressed by the various promoters classified as “high” (the genes arehighly expressed), “stress” (the genes are expressed by activation ofthe promoter or their expression is repressed by exposure to a stressresponse (e.g., change in pH, exposure to bile, oxalate or ethanol aloneor in various combinations) and “sugar” [the genes are expressed in thepresence of sugars such as glucose (glu), fructose (fru), sucrose (suc),trehalose (tre), fructooligosaccharide (fos), raffinose (raf), lactose(lac) and galactose (gal)].

As shown in Table 4, ORFs 1467-1468 are induced in the presence oflactose and galactose. SEQ ID NO:79 comprises the nucleotide sequence ofthe promoter for ORFs 1467-1468 from LacL up through the cre sequencesthat are upstream of LacR. SEQ ID NO:80 also comprises the nucleotidesequence of the promoter for ORFs 1467-1468, but further includes boththe sequences of LacR and the sequences in front of LacR. Accordingly,SEQ ID NO:80 includes the repressor sequence. Our data has demonstratedthat SEQ ID NO:80 allows for the tight transcriptional control (promoteroff) in the absence of the inducing sugar.

Table 5 lists the genes (designated by ORF#, see Table 3) expressed bythe promoters responsive to the stress conditions described herein andthe particular conditions for induction of their expression (pH, bile,oxalate, ethanol). Numbers and shades represent induction of expressionlevels from high (15) to low (2). The conditions for exposure were logphase cells in MRS broth (OD600 nm of 0.3) for 30 mins. FIG. 1 is aschematic of the experimental design of the microarray assays describedherein.

FIG. 2 is an overview of the expression data from GUS (reporter gene)assays that were carried out, investigating gene expression inconstructs including three promoters disclosed herein, as examples. pFOSincludes the sequence of the 502 sugars promoter (SEQ ID NO:72); pTREincludes the sequence of the 1012_sugars promoter (SEQ ID NO:73); pPGMincludes the sequence of the 185 high promoter (SEQ ID NO:6). Thiscovers examples for both the “highly expressed genes category” (PGM-185)and the “inducible by carbohydrates category (FOS-502 and TRE-1012). Thegraph shows that (1) (foremost left) in the presence of FOS as asubstrate, the FOS promoter is inducible (when compared to glucose andfructose); (2) (center) the PGM promoter provides high gene expressionregardless of the conditions tested; (3) (foremost right) the TREpromoter is inducible in the presence of trehalose as as substrate (whencompared to FOS and fructose).

TABLE 1 GUS Activity (pmol MU/ug protein/min) Carbohydrate pFOS pPGMpTRE MRS — 1662.40 — Fructose 17.94 2428.10 147.61 Glucose 13.83 1359.9069.91 FOS 1299.60 3105.30 — Trehalose — 2554.00 833.10

FIG. 3 is a detailed representation (through time) of the pFOS (promoter502_sugars) (SEQ ID NO: 72) data. It shows that this promoter isinducible in the presence of FOS when compared to glucose and fructose.

TABLE 2 Promoters of the present invention and associated genes. SEQ IDExpression/ NO ORF#(s)^(a) Gene(s) controlled Response 1 8 singlestranded DNA High binding protein 2 55 D-lactate dehydrogenase High 3151-154 alkyl phosphonate ABC High transporter 4 169 s-layer protein(slp-A) High 5 175 s-layer protein (slp-B) High 6 185 phosphoglyceratemutase High 7 271 L-lactate dehydrogenase High 8 278 FtsH cell divisionprotein High 9 280-281 lysyl-tRNA synthetase High 10 284-285 RNApolymerase subunits High 11 287-289 30S S12, S7 ribosomal High proteins,elongation factor ef-G 12 290-294 30S S10 and 50S L3, L4, High L23, L2ribosomal proteins 13 295-298 30S S19, S3 and 50S L22 High ribosomalproteins 14 317-318 RNA polymerase High 15 360 50S ribosomal protein L11High 16 369 50S ribosomal protein L1 High 17 452-456 mannose-specificPTS High system component IIC 18 639-640 phosphocarrier protein HPr HighpthP, p-enolpyruvate protein ptI 19 655-656 phosphotransferase systemHigh enzyme II pthA 20 697 transcriptional regulator High ygaP 21 698glyceraldehyde High 3-phosphate dehydrogenase 22 699 3-phosphoglyceratekinase High 23 752 glucose 6-phosphate High isomerase 24 772-779 H⁺ATPase a, c, b chains, High delta, alpha, beta, gamma subunits 25 817isoleucyl-tRNA synthetase High 26 845 translation elongation High factoref-Tu 27 846 trigger factor protein cell High division 28 889phosphoglycerate High dehydratase 29 956-957 phosphofructokinase, Highpyruvate kinase 30 958 Hypothetical protein High 31 968 30S ribosomalprotein S1 High 32 1199-1196 glycyl-tRNA synthetase High alpha, betachains, DNA primase, RNA polymerase sigma factor 33 1204-1201 PhoH,ef-Tu, GTPase High 34 1237-1238 homoserine High O-succinyltransferaseMetA 35 1511 N-acetylglucosamine High kinase 36 1559-1559 FGAM synthesisHigh 37 1599 fructose bisphosphate High aldolase 38 1641glycerol-3-phosphate ABC High transporter 39 1645-1642 ABC sugartransport High 40 1763 oligoendopeptidase F High 41 1779 fructose operonrepresser High 42 1783-1782 ABC transport, ATP- High binding protein,permease 43 1892-1891 adenylosuccinate synthase High and lyase 44 40-38ribonucleotide Stress reductase/cobalamin adenosyltransferase 45 83protease Stress 46 96-97 protease/chaperone, Stress tricaboxylatetransporter 47 166 K⁺ Transporter Stress 48 204 aminopeptidase C Stress49 329 cell division protein ftsK Stress 50 396-395 oxalyl-CoAdecarboxylase Stress 51 397 ABC transporter ATP Stress binding 52405-406 cochaperonin GroES, Stress chaperonin GroEL 53 555myosin-cross-reactive Stress antigen 54 638 ATP-dependent Clp Stressprotease ATP- binding subunit CplE 55 847 clpX Stress 56 9122-oxoglutarate/malate Stress translocator 57 913 Peroxidase Stress 58914 citrate lyase ligase Stress 59 1119 hypothetical inner Stressmembrane protein 60 1234 Cd/Mn transport ATPase Stress or H⁺ ATPase 611246 heat shock protein DnaJ Stress 62 1249-1247 heat-inducible Stresstranscription repressor HrcA, cochaparonin GrpE, Hsp70 cofactor, heatshock protein DnaK 63 1339 Stress 64 1429-1427 transporter-membraneStress protein 65 1432-1430 Stress 66 1433 dihydroxyacetone kinaseStress 67 1446 multidrug resistance Stress protein 68 1683cation-transporting Stress ATPase 69 1910 ATP-dependent protease StressClpE 70 400 Sucrose 6-phosphate Sugar hydrolase ScrB 71 401 PTS systemII ABC ScrA Sugar 72 502-507 ABC transporter substrate- Sugar bindingprotein 73 1012 PTS system beta-glucoside- Sugar specific (trehalose)IIABC component 74 1013-1014 trehalose operon Sugar transcriptionrepressor 75 1442-1437 sugar ABC transporter, Sugar sugar-bindingprotein 76 1459-1457 galactokinase Sugar 77 1463-1462 lactose permeaseSugar 78 1467-1468 beta-galactosidase large Sugar subunit 79 1469UDP-glucose 4-epimerase Sugar 80 1467-1468 beta-galactosidase largeSugar subunit ^(a)ORF# designation is as shown in FIG. 2.

TABLE 3 Sequence/Expression Profile SEQ ID NO: >8_highgtcgtttcgcatatgaaattgataagtatcgtgaaggtacttaccacattatgactttca 1ctgctgacaacgctgacgtagttaacgaatttagccgtttgtcaaagatcgacaacgctatcttgcgttcaatgaccgttaagttagacaagtaattttaatttattgttttcgtgatttaggaaaggatggacaaaggt following gene (8) is consistently highlyexpressed >55_highatcatctctatttgttgcgttgttttttgttatgagtatatattacattttaaatgacaa 2tgtgtcaccatttatttacttgtcttaataaattctttatagtttttcatttgttttcaatgatgtttcacgtgcaactgcttttttagaaaaatattgtttttgtgttttgttgaacaaacggaagtgtataatgagga following gene (55) is consistently highlyexpressed >151_highagcaatttaaaggttttaatgaaaaatttattgttttgggcaagtcttccactcgtgagg 3acgttttttctgttcgtttgattaataatatcgttaacaagcaggcttaattactgatcgtttttgacgacccgtaattaagccttttttgtgggcgaatagtttgttttatcactattttatgttttatggaggacata following genes (151, 152, 153, 154) areconsistently highly expressed >169_highatatgaatcgtggtaagtaataggacgtgcttcaggcgtgttgcctgtacgcatgctgat 4tcttcagcaagactactacctcatgagagttatagactcatggatcttgctttgaagggttttgtacattataggctcctatcacatgctgaacctatggcctattacatttttttatatttcaaggaggaaaagaccac following gene (169) is consistently highlyexpressed >175_highctcccacccaagacaattaataggacgcgcttcaggcgtgttgcctgtacgcatgctgat 5tcttcagcaagactactacctcatgagagttatagactcatggatcttgctttgaagggttttgtacattataggctcctatcacatgctgaacctatggcctattacatttttttatatttcaaggaggaaaagaccac following gene (175) is consistently highlyexpressed >185_highaaaacaactacaaaatatttctttttgtttttcatgatttttacacttctcttagtatgc 6ttttgttataagttagcacaaaaaagcagaaaataaaaagtagaaataaaaaaagatgtttttttgcccatatctctatgaaaaaaactgtgaaatgtgtaaaatatggatgaaacattgaatttaaaaggagatatttc following gene (185) is consistently highlyexpressed >271_highaccagtattatgtttggtcttatcatatttttgacccggattacccaaacctgcaattat 7cttcatcttatttacccctcattaataataatctcaactataatagcacaaacacaaaataataattttattaatgctcttcaacatggtataattttctttgttaaaattatcactaataaaaaaggagacttattgtt following gene (271) is consistently highlyexpressed >278_hightgtttagcaatttatgctgatcgagaaccaattttcgttgaaaatacgtatcaaaatcaa 8aattggataaaaaatggcaaacattattttctatatgctaattaatttatcagtaaatatagttgaaaatattagtggtcggaacttgttttgtgataaaattttaaacgtataacttaaagactttgcggaggtttttt following gene (278) is consistently highlyexpressed >280-281_highagatatgatcaatgaagatcatggagcagaacttatttgcaacttctgtggtaacaaata 9ccattacactgaagatgaattgaaagagattttagctaagaaaaaagacgataaagattattaattaaatttaaagaggcctaaggttttaacctttagggcttttttgatattataataaagtattttgaaaggatgat following genes (280 and 281) are consistentlyhighly expressed >284-285_highaaaaataaaaaaatattatacaatttttgctgatttaaaaagactgagattcaggatttt 10gctgatctattgtccagcaaaatgataaggacaaaaacgacacttgttgtttttgtcttttttatgcctaaaattgcggttttttgaatttgtaacagaaatgtaatatttgctttcttagacagaaaggatgtttttcc following genes (284-285) are consistently highlyexpressed >287-289_highaattaagtaaaaaatatattgagttcaaaaaatcacctcattgtttattacgcaaaattc 11aaaaaattctttttaaaaagtttgatttctattaaaaaccgagtacaatagtctttgtatgttttgaacagtctattcgcgagtataaaaagaaactcccggatgtgcgaacaaaatagtatttttaggaggaaaaatta following genes (287, 288, 289) are consistentlyhighly expressed >290-294_highttgtaacccttgatatttaaggacataccaagtacaatagtctttgtgcttaaggggcga 12ttgcgccctaagcgagtaatattgttgtagagcgttgacgcaaaaggttgcggcacgccaggctgcattgccacagtggcgtgcggggaatttttgccgagcgagtcatcttttaaagaagacgttaaggaggtaattta following gene (290, 291, 292, 293 and 294) areconsistently highly expressed >295-298_hightaaggctccagttggtcgtccacaacctatgactccatggggtaagaaggctcgtggtac 13taagactagagatgtcaagaaggctagcgagaagttaatcattcgtcaccgtaagggtagcaagtaatagaaggagggttaattaatgagccgtagtattaaaaaaggtccttttgctgatgcgtcattgttaaagaagg following genes (295, 296, 297 and 298) areconsistently highly expressed >317-318_highaacatgtagaagtttctgttaaaggtcctggtgctggtcgtgaatctgctattagatcac 14ttcaagcaactggtcttgaaattactgcaattcgtgacgttacgccagttccccacaatggttccagaccaccaaaacgtcgtcgtgcttaattttgtccatgatattataggacgttacgttttgaaaggggcccagta following genes (317, 318) are consistently highlyexpressed >360_highagaccaagtcaaggaaattgctgagactaagatgaaagaccttaacgctgctgatattga 15agctgctatgcgcatggttgaaggtaccgctagaagtatgggtatcgaagtcgaagactaatcctgttatttagttaacacattaggtgggagagttaagagaagctcgtttgaccacatatacaaggaggaattcacac following gene (360) is consistently highlyexpressed >369_hightttatccttgctatctttgataatgcctgctacaatagttaattgtaaattctacctaag 16actcgggtggcatgacgcctcaaaatcccgccgaggccagaagataatgaagatttttatgctctatgtctttcggcatggagtttttgctttaaaaggccttatagaatttattaatgcgattatggaggtgaaattaa following gene (369) is consistently highlyexpressed >452-456_highaaaatccctttttatgacaaaataaaagggatttttttattagactaatttgagcatttg 17gcttgaaccgcaaggcttttcgtcttatttgaaatttatttatattgtatgaaattatttccaaaaagtactttgtaaaagtgtgtatttatcgtataataaaagcggattcatttttttgatctagaggaggaaattac following genes (452, 455, 456) are consistentlyhighly expressed >639-640_highgaaattatggcaaacgacaatatattaccggcagggccgaaagaggcggatctatcgtct 18atactgcgacaaataccgatgattgaatgatgtaaactgttacattattgttgtctaaactgtaaaaacatgataatctattactcgaatgggtatttattaccagtttaattttttttaatttaaaggagatattcata following genes (639-640) are consistently highlyexpressed >655-656_highttaataacattttcaatactgtgccgctgaatggggtagactggttgtttctcttccttc 19ttcctattccgctagttctattagatgaagtaagaaagtggttaatgtattacaacaaaaatattaattaatttttatgtaacttaagtgtttaactgacctttcttatgctagaattgactttaaggagatatataatt following genes (655, 656) are consistently highlyexpressed >697_highaattatttcactcttcttaggatatttttaaaatagcacatctttttcttgaattactaa 20aaataccttgttatactaacagtgtcgattgggaaatgtatgaattgaagaatcgtacgtttctcttatatttttaagtaatctgggacagaaagtgacacaggggtggtcaatatacgtcccagggaaaggagggaacg following gene (697) is consistently highlyexpressed >698_highaatctatataaaataccccacatatttgcctttgcttgcggtgctaaaaaagctaaagca 21attaaagcatatatgcccaatgcacctcatcaaacctggttaattactgatgaaggggcctcaaatatgattttaaaggggaaatgaaatcccgtttaaaataaattgttgtttatagttcttaaggaggactttaggtt following gene (698) is consistently highlyexpressed >699_highttagttaagactgttgcttggtacgacaatgaatactcattcacttgccaaatggttcgt 22actttgttacactttgctactctttaatcattaattttaattaactgattatagttaagtggtaatcgagaaggcggagggagattcttccttccgcctttttttgaagaaaaaataaatattttttgaggagaatatta following gene (699) is consistently highlyexpressed >752_highgaaacgctacagtttttattaatgacaggtgttagtgatattgatgacgtgttttttaac 23acttgtggcgctattttaggctatttaatatatattcttttcaaaaaaaggtgaatgcgcttataattggtactggtattcaagaaataagattgttaaaataaaaatgttaaaatttttaatagttaggaagcagattt following gene (752) is consistently highlyexpressed >772-779_highgatgataaattgctggacaatggttacattttccctggtttgggagatgccggtgacaga 24ctcttcggtactaagtaaacaccttttcacaaaaaatatttactctaatgcgctttcattttacacaaagaagatatttggtgttaagatgatttacgtgttcgagttttattcaacacgagaagggaggtcacgaagta following genes (772, 773, 774, 775, 776, 777, 778,779) are consistently highly expressed >817_hightagtggcgattcagcgagttagagatggtgtgagactaacataagtgcccaaaagttgat 25cggctgccatattgatctaagcgttttttgcacgttacgcaaaagtaagtggaattctttttagaattcaatttaggtggtaccacgattaacctcgtcctaatttggacgaggttttctttttagaaaggattttatta following gene (817) is consistently highlyexpressed >845_hightaccaaattaaaataataagcaaaaaaggtttacattttcgaactatttagtataattag 26caaaggatattttcgttaggcatatcgcttaatcttttttactaggcatttgccgaagaaagtagtacaatattcaacagagaattatcctttaacttatctcaacggacttcttgcaaatttacaggagggtcatttta following gene (845) is consistently highlyexpressed >846_highccgtgaaggtggtcgtaccgttggtgtcggtcaagttactgaaatccttgactaatttct 27aacgatatagttaaaaaagatgcacttcttcactggagcgcatcttttttcttttatatttgttttttgtgctagtttaaggtaagataacttagtatgcaagaagcaaactcaaaattgacattggaggtattttatta following gene (846) is consistently highlyexpressed >889_highagttacgttatacatatattatagctctttgatatagcattttttactgtgctttactat 28tttttaaaatgtaaaccgctttcatatgtttacacgatcacaaagttaggctaaaatttgtgttgtaaagcggagcaaaaattgttccgtatggcatgcaaaatttttgttacatgccataatttttgaggaggtttata following gene (889) is consistently highlyexpressed >956-957_highaaccaatttacgtaaagtaaactttaaagaataattgtctactttaaagaattgaattat 29caatatatgtaagtgctaacataaactctgaagtgagaaacaataaattagcccaatttttgtgagatttttggtctaaaaaatgttaatatttacttgatgtgagaaattacacaaaataatcatgatgaggtgaattc following genes (956, 957) are consistently highlyexpressed >958_highagatttctgacggttcaactattactgttgatgcttgtcgtggtgctatttaccaaggtg 30aaatctcaaacctttaataatatataaataaaacagattagctaatcaaaaaatagtcagcttttgagctggctattttattttgttcgaatatctcttatacttatatataaagaatatgtaaagtaggagatttttta following gene (958) is consistently highlyexpressed >968_highctcatcgcaaggtttcaccactaaaaaaggcagatgatgctattgaaattgatactacaa 31atatgtcaattgaccaggttgtagatgcaattttagctaaaatcaaagaaaattaaaaaatttttttaaaaaaacagcacaaaatagtagaaaaatatcacagtttcctttaaaatgggacatgatattgggaggtacat following gene (968) is consistently highlyexpressed >1199-1196_highataatgaggattagaaaagtactagttcagtgaatgtcgtttggtgagaggacatctagg 32aaaaggcccctctagtcatactcaattaagtgcaggaagaagacttcctgaattagggtggaaccgcgagatatttcgtccctatgcaaaattttgcataggctttttttatggcccagtgcaggaggagaataaggaaa following genes (1199, 1198, 1197, 1196) areconsistently highly expressed >1204-11201_hightaacaaaatttaaaaatatttagtagtcataaaataagataatctggtattaagtattta 33agccttgaataaaggatacaataatttagttttcaataaaaatattccatataatagtaaataatcaatagttttattttagttatgtagatagtttgttataatactattgggtttttaatagaaagaaggataccaga following genes (1204, 1203, 1202, 1201) areconsistently highly expressed >1237-1238_highactaatgaatatttcgcccaacaatcatcttggttgaagttcaagcaatacttctctaga 34ctactttcacctattttttaataatatatttcaaactgacaaaatattttgtcagttttttctttaagtgtttttcctttacttaatttttaataagctgtataattaacccaactattaataagtaaggaggtaaaatc following genes (1237, 1238) are consistentlyhighly expressed >1511_hightacccttgtttatatcccgtggatattcttagttggtatcctaattagcctcttaactat 35tataattttattaagaattggataaaaagcaggcatgataacgctaacagcaaaaataatgctggaccaaccacccaaaaatgtaataaaatgtatacgttatcattcggataaattaataacagaaagaagatttgaat following gene (1511) is consistently highlyexpressed >1559-1552_highctttgaaaaagagagctataaagctctctttttttgttcaattcttacaaaacacgaacg 36attatttatactatcatttttaatattcaataaatcattgacattacagacacttattgataatattgttagcataaaagtgaacgaataattattcgcttgccagaaatgttcgtgttttttaccaaggagaaagaaaa following genes (1559, 1558, 1557, 1556, 1554,1553, 1552) are consistently highly expressed >1599_highttggtgctcgccttgctttagttcaggctacatcaatcgttttgactgaatcacttagac 37ttttaggtgtaaatgctcctaaggaaatgtaaagatttcaatgaaaagtaaaaaatagcgcttacattttgtgaaaaattgttcataatcgaattaataaggtacaatatgcatgtaagatatttaggaggtatttttta following gene (1599) is consistently highlyexpressed >1641_highcatgctgatgaaggagaactcaaagaaattattggcgggattcagccagctgttttggta 38ccggtgcacacactgcatccggagctggaagagaatccatttggagaacggattttacttaaacgtggccaaactgtcacgctttagtgaatcaaaaaatatattgttgtttagttttattttttaggaggatttatcta following gene (1641) is consistently highlyexpressed please note the RBS is one base shorter than chat shown in thegenome file >1645-1642_highaaatacgaacaaaaagacaaaaaagtagtttttgatttataaaatacgaaccagatacga 39atgctaagtgaaaaatatttcatcaataagggataactacgaattttttacatgaaatatttgtgatttttgtccatatagcttagaattaataaggaattttataaaaataaatcaatatatagtggtgtgtgaaactt following gene (1645, 1644, 1643, 1642) areconsistently highly expressed Please note a RBS was not found >1763_hightaatcttctctacgtttggaatttggatccattctttgtatcgtttcccttcaaaattaa 40tacaagtttatttgtatcacttttaatctctatgataaaataaaattatcgataattaataataacttagtttttgagttaaattctacatcgaaatgcatctttaacaaagatggaatatttttcaggaggaaacaaat following gene (1763) is consistently highlyexpressed >1779_highattcttagtcaaaaccaaaaaaatgactaagaataattcaaaatgacgaagaaaagatgt 41cgtttcaatcaaaaaacggcatcttttttgcatataaatgaattttattgaatgataataaataaaaatgacgctttttgaagaaaaatggttgattttgatggagaaagcgaatacaatgtttatcgaggtgagaaata Also note the RBS does not appear on the genomefile >1783-1782_highggtaatgcgacacaaaacatcggatggttatcaactaagatttactcgtaaatctaccaa 42agtatatcctcatttttggcaagcatattggtgtcaagcgttgctgaatattttggatgtatgtgatattttatttctatataattaaatttagatactaaaaatatcgaatcaatatcaaaaaagttgaggaaaaaatc following genes (1783, 1782) are consistentlyhighly expressed please note the RBS is one base shorter than that ofthe genome file >1892-1891_highaatttgatttgctccctttattttctgcttaccaaacgagaactactatatttgataaaa 43gtatttttgtcaatataaaaatcgaactatgaaataaattaaaaataaaataattcggtttttgcattgactaataattaacaaattgctagactatcatacgtaatatttatagagattttttatgaggtgaatttcaa following genes (1892, 1891) are consistentlyhighly expressed >40-38_stressacttgtaggacaaactgattgtgaaggcggggcctgcccaatcaaataatttacatggag 44gaaaaaatgaagaacaagcataaatttaatttattattttcaatcgttgcctttttggcttatttttaacgggaagttcaaatagtaattcatctgctacaaaaaatactgctaaaaatcaaattacagtcaactatact following genes (40, 39, 38) were induced over 2fold in the presence of oxalate >83_stresscagaatatttggctcgtgaaacggcaaaagagatgctgattgatggggaggcaaatatta 45acagtgatttaaaaatcattgatacagagccgaatcacccaacaaaattaattgaaatttagcagttttagcaccttttgttcatataattttcaaattttatctgtatgatattaggtaatatgaggggagagttaagt following gene (83) was induced over 2 fold in thepresence of ethanol and over 4 fold a the presence of bile >96-97_stressgtaagtcgcaaaaagttctttattcaatggactatcatgcaattcgctgggttaatcatt 46ttgaccttagttggtttaggactactaatgtttagactttaaattttgttcaagaatgcttttatagcattcttttttattgctctaaagcctataaaaattataaaattatataaatactttttatggaggattctatc following genes (97, 96) were repressed over 4 foldin the presence of pH 4.5 >166_stressgaagaaaatgggtgtatcaagaaaatgtcaacatggcattttctttttttatatttttta 47tactagctacaatttattttgtgggagatttttgataatgaataataagtccaaacgtatgagtgcggctggccttcttatcgccatcggtattgtttatggtgatattggtactagtccactttatgttatgaagtcaa following gene (166) was repressed over 2 fold inthe presence of bile and ethanol, and repressed over 3 fold in thepresence of oxalate and pH 4.5 >204_stressatcaccctaccaaagcaaactgctggggatgatgataattctatccagattgattaaatt 48attagattattgcaagaagtctgattaatttaaatggataattctctaaaacgggttcaatgattgaacccgtttttgttttggcttaaaatagtagttaatttaagaaaagattaaaaatgagaaaaggagatttttta following gene (204) was induced over 2 fold in thepresence of pH 5.5 >329_stressattgatggtaaacccaccaatgaaataaatggttatcaagcttggtttgtcgcagaaggt 49actgaaccttttaaagttaaatttactaaaagagtcagtcttccaaaaatgttaaatcaaatttcattaacaaatcttgaggcttgtgaagttggatcaaatgtgtattttaaagcaacagatctcgaggtgattaacta following gene (329) was induced over 4 fold in thepresence of ethanol >396-395_stressgcattggataattttgaataatacagtaaaaagaatacttatttatttatataaaaaagt 50attctttttatttgtgtacgcatattataaataacacaacttattattcaatttgcttgtatcttttttttaagaggtgtatcttgaacttgaaatgcaagatgaaagcatttttgggatttttgaaagaaggttttttc following genes (396, 395) were induced over 3 foldin the presence of pH 5.5 >397_stressatttttactttctccatagttgatcctccaaacgtatctcaagtttgtgaattcacaatc 51aagcatttctatcataactgttaatataccatttacgcaagtgccagcttcacaaatatacttttttcacatatataattcaaaatagtgagctcagttaattcacatcctgtgataaaatattggttaggtgaaaaatt following gene (397) was induced over 3 fold in thepresence of ethanol >405-406 stressgatctaaatattcaacatgttaaaactgaataaaaacacaattagcacttttttataaag 52agtgctaattttttcttgcttttttttagtaaacgggttattatcatatttgtaagttagcacttaactaaaaggagtgctaacaatcaaaaatgattataaataataatgaagaaaataaattataagggggactaaac following genes (405, 406) were induced over 5 foldin the presence of pH 4.5 and bile, and induced over 10 fold in thepresence of ethanol >555_stressgtaatagagatatctatgtaaggctttttttgtagtaatgaaaataaagttttttcgatt 53tgttgctgagttcgcatgcttttcatgttcatagtgtattatcccttatatttgtattagttgacatatgaaagcacttacactatcattatagttgtaaatagttgcagatgtgacgatttttgaaagaagtgtaaatt following gene (555) was induced over 2 fold in thepresence of pH 4.5 and bile, and induced over 12 fold in the presence ofpH 4.5 >638_stressataatccacaaatatcaccacactttttaaattttataatttttcttctttttattctac 54tctttacactaaattttctaaaatattaacattttatttaattcttacaaaaaataagttaaattggcgcttagcacttgactaccaagagtgctaaatatataattttggtacagtttaattgaaaggcggtaatatat following gene (638) was induced over 5 fold in thepresence of bile and induced over 12 fold in the presence ofethanol >847_stressttactgacaatgctaagcaagttgctaagtcaaaacttgaagcaaaagattcagacgata 55aagaaagcaagtaagactaatttacttatttcttaaaaggagcggctttaggttgctttttttaatgttcaagcttaatatttactaatattagttaatttatgataatctaattttggtagatataggaggaaaagtta following gene (847) was repressed over 2 fold inthe presence of oxalate, bile and ethanol, and repressed over 4 fold inthe presence of pH 4.5 >912_stressttattgtgagcttttttagttaataaataataacaagtatagatttgaacatatttcgta 56agatattttacttttaaaatgatgaaaaaacattatttattttgaaattatttaaaaacaaaataaaaagtatataatgagtatgtgaaaaaattcattttatattgattgctttgataaaactaagcaggggaaggaaa following gene (912) was induced over 2 fold in thepresence of pH 4.5 and pH 5.5 >913_stressggcgacaacaggctatgtaaaacaaagtgaatggtggaagatgaactttattttagggct 57tatttacatggtgatatttggtatagtaggaactatttggatgaaaattattggtatttggtaaaaataaaggcaatctgatttcatagattgccttttttgcgtgataattgaggggtaggaatagaaagaagaaaaag following gene (913) was induced over 3 fold in thepresence of pH 5.5 >914_stressgaaaatatacaaactgcaattattcctgaatgcggtcatctacctcaggcggagcgacca 58gatgaagtatataaaattattagtgattttttgaaaaatttaaaaaactagttctaaaattgaaataattaaactgcaggagtacactgttcttgtgaaaaagattactttttattaatgcttagtaaggtggcacattt following gene (914) was induced over 4 fold in thepresence of pH 5.5 >1119_stressattatctctaactaacttaattatagtattttttaagaaatgttaaagaaagagacacaa 59tgtcactaatacgcaaatattgtgatattatgagcaatgtaatcaaacaaagttcggggactttgtaaagcaactttttacatttggaggttttattattggagattgtcaaaactaaatcatttagattagctgttgct following gene (1119) was induced over 3 fold inthe presence of bile, induced over 5 fold in the presence or oxalate andinduced over 7 fold in the presence of ethanol >1234_stressatcttttagcagcagcagtaatttgcttttcttcagctaccactaagaaataatgtaatt 60gtttaatattcatcaaatatatcctttttattttcatgagcagtgatattaaaaaaattaatatcatatattttattttagtattttaatctaagcaatttatatgctaatttagttaaagaagatttggagggagaaaa the following gene (1234) was induced over 9 foldin the presence of oxalate >1246_stressaatggtggtgctcaaggtgcagctggtcaagcaggtcctcaaggcggcaacccaaatgat 61ggtaacaatggtggtgcccaagatggtgaattccataaggtagatcctaacaagtaatgggtttataattaaacaaaaagagaaaagaactacccattgagtagttcttttcttttgaaaacgataaggagttcaattgc the following gene (1246) was induced over 2 foldin the presence of bile and ethanol >1249-1247_stressagcaagttcaaccagcgatgattttagttgaacatgatgaatactttattgaacgagtag 62ctaatcaaagaattactttaaacttgaagaaaaaagtttaaaataaattagcactcaggttgcattattgctaatttctagtataatataatctgttagcacctgatagatgtgagtgctaaaagtgagggcgatatata the following genes (1249, 1248, 1247) were inducedover 2 fold in the presence of bile and ethanol, repressed over 2 foldin the presence of oxalate, and 1249 was also induced over 2 fold in thepresence of pH 4.5 >1339_stresstcttcatctaaaggatatcgttcatttgaacgggcactttacagagctgaaaatggctta 63ccggcatatgagggtactcaatcaattgaatataaacaggaagaaattaaataatttattaatattattttaatttgttgtggcgagatatattttttcgttaaaatagaattactaactaaagaaaggacgcttactg following gene (1339) was induced over 2 fold in thepresence of oxalate and bile, and induced over 4 fold in the presence ofethanol >1429-1427_stressgtgaagatgaatctcacaattctaaaaaaggtggctttggtattggattagctatggctc 64aagaattaattcatactttccacggtaaaatttcagtaaatcatagagaagaaaatatcgtttttagtgttagtctaaaaattgtcaaatagatgttcttgatagttgtataatttcaattaaagaatcgaggaattatt following genes (1429, 1428, 1427) were inducedover 2 fold in the presence of bile >1432-1430_stressatcgcttacaaatagattataacgcaataacttataaatttaaaaacatatgacatgttg 65tcatatgtttcattaagtaaacgtgatttttacaattttaaaataattttatagcaagttataacttttataatattcctgttactttcaaagaaaaatcaaaaatcattgctataatggcgtaaacgaaagaaaggaca following genes (1432, 1431, 1430) were inducedover 2 fold in the presence of bile >1433_stresstatagcaatgatttttgatttttctttgaaagtaacaggaatattataaaagttataact 66tgctataaaattattttaaaattgtaaaaatcacgtttacttaatgaaacatatgacaacatgtcatatgtttttaaatttataagttattgcgttataatctatttgtaagcgattgcatttttgcaaaaggagaaatt following gene (1433) was induced over 2 fold inthe presence of bile, and induced over 5 fold in the presence of pH4.5 >1446_stresscaaaaagctggtgtaatttatttttctttatattttccattatctctgcctcactaatta 67aaattaattatattaatttatgttaaatttttcaactttagtgtcatattatgtatcatatttgtaagattatttgacacagattaaaattaggactatattagttaacgatcttaattttcacaaaagggggatgacac following gene (1446) was induced over 7 fold inthe presence of bile, and repressed over 2 fold in the presence of pH4.5 >1683_stressgaagttgataaacctcaattagtataattgacactatgtcactaatgcgctattatatta 68gataatcaatatattgagaagcgcctatgacgctgccaatacacaaatgaaaactgaacaagtttctcaaatggggaatggcttatgtaagtaggctgttctctatttttttattttatgaaaggagtggtatatccgat following gene (1683) was induced over 3 fold inthe presence of bile and ethanol >1910_stressgtgaagcattacgagcgctttaatatcaagcgattaaggccaattttatatttttaatca 69caataaagaataaaaatgtggaaaaagttcaaaataatacttgcaatctgtggataacatgttatacttataaatgtaaagaattagcactcaacgcactagagtgctaatagacttaaattgattgggagtcgtttatat following gene (1910) was induced over 2 fold inthe presence of bile, and induced over 15 fold in the presence of bile,and induced over 15 fold in the presence of ethanol >400_sugaraattcactatttatgataacgtattcaaaaaatatgtcaatcgtttgacacatttttttg 70aatttattttttattaatacttttcttatggtccaataaggcaagggtagtcaaatataatatgataaacgtttgacacatttttcataatctactagaattaatattaaagataacgcttacatggaggcttttttatt following gene (400) was induced in the presence ofsucrose >401_sugartattaattctagtagattatgaaaaatgtgtcaaacgtttatcatattatatttgactac 71ccttgccttattggaccataagaaaagtattaataaaaaataaattcaaaaaaatgtgtcaaacgattgacatattttttgaatacgttatcataaatagtgaattgagaataaaagcgtttacataggaggaaacaaat following gene (401) was induced in the presence ofsucrose >502-507_sugaraactgttgacaagttgtgaaagcgatattatcatttaattgtaaattgaaaacgtttcca 72aagtgttcaaatagttttttgctaaataattatttttttgtagcgaaatagaaacgtttcaattaatttaaaacaattagatcttagtaggaaaccttttaatttttgtgcaaaattgaaacgtttcaaaaggaggaaaa following genes (502, 503, 504, 505, 506, 507) wereinduced in the presence of FOS >1012_sugarctgattttgattccgtcatttatgtctttcttttctttgtacatttattatatttataaa 73tgtatagacaagtaaagcataatttaagttactataaagtaaatattgtgattgctttcaaaaaatatattgacaacttgtatatacaagtttaatataatagctaaatctaatgaaaacgctttatacaggagaaaaaca following gene (1012) was induced in the presenceof trehalose >1013_sugarttcattgttttcattcattgtttttctcctgtataaagcgttttcattagatttagctat 74tatattaaacttgtatatacaagttgtcaatatattttttgaaagcgatcacaatatttactttatagtaacttaaattatgctttacttgtctatacatttatgaatataataaatgtacaaagaaaggaaagacataa following genes (1013, 1014) were induced in thepresence of trehalose >1442-1437_sugargtattctaacatttgcttttattgcttacaatacaccgattagtaaattaaatatgtcaa 75aatgtttataaggccaaatgacaataatgctaatgaaaatactatggtttacatacatagaatacgcaataattaaatatgtaatttatgaaagcgcttaaaattgaatgctatttatttagttattgaggagtgatctt following genes (1442, 1441, 1440, 1439, 1438,1437) were induced in the presence of raffinos >1459-1457_sugarttggtatcgtgatgtgataaaagaaaatggacaaaatttaaaataattagtttaaaaaag 76aaaatattcttacagaatgtttccttttttattatataaaattaaataatttatttatttgagtaaaccatttaccaaaaacaaataagagtatatactattatctgaaaacgattacagtaaaaattgaggtaaaaacg following genes (1459, 1458, 1457) were induced inthe presence of lactose and galactose >1463-1462_sugarataaaaagaaataaagacaacggggctggctaagcccttaaactgtaagagctggtcaat 77gtgattactcccaagtggaatatcagaatactagtgaagacgacagtaagtgaaacaaagaaaggaaaaatatatctttctgatatgtagaaaattcgtcttcttctacatatttccatgttttatatagcaggaatatt following genes (1463, 1362) were induced in thepresence of lactose and galactose >1467-1468_sugaracttacttacgtttattatacaaaatatttactcaattccaataaatattaattttagca 78aaaacaaattttttaagaatcttcgtaataaatattttactgtttttagataaatattttattttattggttaattttttatttggtgatataataaaagcgttttcaaaaataatttattatagaaatcaggtattagt following genes (1467, 1468) were induced in thepresence of lactose and galactose >1469_sugartagttattgctggagctgtgcgcggcgttggtggtatcgacagctggggtgctgatgttg 79aaaagcaatatcacattaatcctgaaaaagactacgaattttctttcaatcttaattaaatattttatcaataatagtaaatgttttactgatttatgtgttataatgtaatcgatttcaagaaaacaaaggagtaaaca following gene (1469) was induced in the presenteof lactose and galactose >1467-1468_sugar gtggcaggtg aataacccgatttttgtgca atctctttaa tagttgtcat agttaatttc 80 ttttcttttt aaaaaacttacttacgttta ttatacaaaa tatttactca attccaataa atattaattt tagcaaaaacaaatttttta agaatcttcg taataaatat tttactgttt 1 ttagataaat attttattttattggttaat tttttatttg gtgatataat aaaagcgttt tcaaaaataa tttattatagaaatcaggta ttagtcaagc aaacataaaa tggcttg following genes (1467, 1468)were induced in the presence of lactose and galactose, promoter sequencecomprises the repressor sequence which allows for tight transcriptionalregulation

TABLE 4 CRE ELEMENTS IN PROMOTERS REGULATING SUGAR UTILIZATION SEQ IDNO: La400 cre1 TGaTaaaCGtttgaCA  −72 bp 90 cre2  AGataaCGcttaCA  −17 bp91 La401 cre1  TGaataCGttatCA  −48 bp 92 cre2  TAaaagCGtttaCA  −17 bp 9394 La452 cre1  TAaaagCGgattCA  −27 bp 95 La502 cre1  TGaaagCGatatTA −172bp 96 cre2  TGaaaaCGtttcCA −140 bp 97 cre3  TAgaaaCGtttcAA  −78 bp 98cre4  TTcaaaCGtttcAA  −14 bp 99 La1012 cre1  TGtgatCGctttCA  −82 bp 100101 cre2  TGaaaaCGctttAT  −15 bp 102 La1013 cre1  ATaaagCGttttCA −155 bp103 cre2  TGaaagCGatcaCA  −88 bp 104 La1442 cre1  AGaataCGcaatAA  −69 bp105 cre2  TGaaagCGcttaAA  −38 bp 106 La1459 cre1  TGaaaaCGattaCA  −27 bp107 108 La1463 cre1  AAaattCGtcttCT  −36 bp 109 La1467 cre1 TAaaagCGttttCA  −32 bp 110 La1469 cr31  TGtaatCGatttCA  −21 bp 111 43HIGH promoters involved in the expression of 88 genes 26 STRESSpromoters involved in he expression of 37 genes 10 SUGAR promotersinvolved in the expression of 25 genes 79 TOTAL promoters involved inthe expression of 150 genes

TABLE 5

The foregoing is considered as illustrative only of the principles ofthe invention. Further, since numerous modifications and changes willreadily occur to those skilled in the art, it is not desired to limitthe invention to the exact construction and operation shown anddescribed herein. Therefore, accordingly, all suitable modifications andequivalents fall within the scope of the invention.

All publications, patent applications, patents and other referencescited herein are incorporated by reference in their entireties for theteachings relevant to the sentence and/or paragraph in which thereference is presented.

1. An isolated nucleic acid molecule having promoter activity, whereinthe nucleic acid molecule is selected from the group consisting of: (a)a nucleic acid molecule comprising a nucleotide sequence as set forth inany one of SEQ ID NOS:65 or 73; (b) a nucleic acid molecule thathybridizes to the complement of the nucleic acid of (a) under stringentconditions and has promoter activity, wherein said stringent conditionscomprise 6×SSC, 0.1% SDS at about 45° C., followed by a wash of 0.2×SSCand 0.1% SDS at 50-65° C.; and (c) a nucleic acid molecule having atleast 95% identity to the nucleotide sequence set forth in any one ofSEQ ID NOS:65 or 73 and has promoter activity.
 2. The nucleic acidmolecule of claim 1 comprising a nucleotide sequence as set forth in anyone of SEQ ID NOS:65 or
 73. 3. A recombinant nucleic acid moleculecomprising the isolated nucleic acid molecule according to claim 1operably associated with a heterologous nucleic acid of interest.
 4. Therecombinant nucleic acid molecule according to claim 3, wherein saidheterologous nucleic acid of interest encodes a protein or peptide. 5.The recombinant nucleic acid molecule according to claim 3, wherein saidheterologous nucleic acid of interest encodes an antisenseoligonucleotide.
 6. The recombinant nucleic acid molecule according toclaim 3, wherein said heterologous nucleic acid of interest encodes aribozyme or interfering RNA.
 7. A plasmid comprising the nucleic acidmolecule according to any of claim
 1. 8. A method of transforming a cellcomprising introducing the nucleic acid molecule according to claim 1into a cell.
 9. A cell comprising a heterologous nucleic acid moleculeaccording to claim
 1. 10. A cell comprising the plasmid according toclaim
 7. 11. The cell according to claim 9, wherein said cell is alactic acid producing bacterial cell.
 12. The cell according to claim 9,wherein said cell is selected from the group consisting of a cell of agram positive bacterium, a lactic acid bacteria, Lactobacillusacidophilus, Lactococcus lactis or Lactobacillus gasserei.
 13. A methodof controlling the transcription of a nucleic acid of interest in a cellcomprising: (a) providing or maintaining the cell under non-inducingconditions, said cell comprising the recombinant nucleic acid moleculeof claim 3, and (b) subjecting said cell to inducing conditions wherebytranscription of said nucleic acid of interest is increased as comparedto the level of transcription of said nucleic acid of interest undernon-inducing conditions.
 14. The method according to claim 13, whereinsaid nucleic acid of interest encodes a protein or a peptide.
 15. Themethod according to claim 13, wherein said nucleic acid of interestencodes an antisense oligonucleotide or a ribozyme.
 16. The methodaccording to claim 13, wherein said inducing conditions described in (b)are produced by an increase or decrease in the concentration of a sugarin said cell relative to the concentration of said sugar in said cellunder non-inducing conditions.
 17. The method according to claim 13,wherein said inducing conditions described in (b) are produced by thepresence of a stress response protein.
 18. A method to express anucleotide sequence of interest in a cell comprising introducing intosaid cell the recombinant nucleic acid molecule of claim
 3. 19. A methodto express a nucleotide sequence of interest in a cell comprisingintroducing into said cell the nucleic aid molecule of claim 1, whereinsaid nucleic acid molecule is heterologous to said cell and is operablylinked to said nucleotide sequence of interest.
 20. An isolated nucleicacid molecule comprising a nucleotide sequence having at least 95%sequence identity to SEQ ID NO:6, wherein said nucleotide sequence haspromoter activity.
 21. A method to express a nucleotide sequence ofinterest in a cell comprising introducing into said cell a recombinantnucleic aid molecule comprising a polynucleotide having at least 95%sequence identity to SEQ ID NO:6 or comprising SEQ ID NO:6, wherein saidpolynucleotide has promoter activity and is operably linked to thenucleotide sequence of interest.